Eunicellin-Based Diterpenoids from the Formosan Soft Coral Klyxum

Sep 10, 2013 - Department of Medical Research, China Medical University Hospital, China ... Graduate Institute of Natural Products, Kaohsiung Medical ...
3 downloads 0 Views 987KB Size
Download PDF
Article pubs.acs.org/jnp

Eunicellin-Based Diterpenoids from the Formosan Soft Coral Klyxum molle with Inhibitory Activity on Superoxide Generation and Elastase Release by Neutrophils Ming-Chang Lin,†,▽ Bo-Wei Chen,†,▽ Chiung-Yao Huang,† Chang-Feng Dai,‡ Tsong-Long Hwang,§ and Jyh-Horng Sheu*,†,⊥,∥ †

Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan, Republic of China Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan, Republic of China § Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan, Republic of China ⊥ Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan, Republic of China ∥ Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan, Republic of China ‡

S Supporting Information *

ABSTRACT: Eleven new eunicellin-based diterpenoids possessing a cladiellane skeleton with a C-2, C-9 ether bridge, klymollins I−S (1−11), have been isolated from the EtOAc extract of the soft coral Klyxum molle from Taiwan waters. The structures of compounds 1−11 were elucidated by extensive spectroscopic analysis, including 2D NMR spectroscopy (COSY, HSQC, HMBC, and NOESY). Compound 5 exhibited cytotoxicity toward several cancer cell lines. Compound 5 is the first eunicellin-based metabolite bearing a phenyl group and displays significant inhibition of both superoxide anion generation and elastase release in N-formylmethionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced human neutrophils.

O

ur previous chemical investigations of soft corals of the genus Klyxum and Cladiella have afforded several eunicellin-based diterpenoids, of which some have exhibited interesting bioactivities.1−11 In order to discover novel and bioactive substances from marine invertebrates, the chemical constituents of the soft coral Klyxum molle, which has been previously investigated as Alcyonium molle,12 were further studied. During the course of our initial investigation of new substances from K. molle, eight eunicellin-type natural products, klymollins A−H, were discovered.9 In this paper, we report our continuing chemical investigation of this organism. This study led to the isolation, structure determination, and biological activity of 11 additional eunicellin metabolites, klymollins I−S (1−11), from more polar fractions of the soft coral extract. The structures of 1−11 were established by extensive spectroscopic analysis, including 2D NMR (COSY, HSQC, HMBC, and NOESY) spectroscopy. The cytotoxicies of metabolites 1−11 against a variety of human tumor cell lines including human erythro myeloblastoid leukemia (K562), human acute lymphoblastic leukemia (Molt-4), and human breast carcinoma (T47D) were investigated. The abilities of 1−11 to inhibit superoxide anion generation and elastase release in fMLP/CBinduced human neutrophils were also evaluated. © 2013 American Chemical Society and American Society of Pharmacognosy

Received: May 8, 2013 Published: September 10, 2013 1661

dx.doi.org/10.1021/np400372v | J. Nat. Prod. 2013, 76, 1661−1667

Journal of Natural Products

Article

Table 1. 13C NMR Data for Compounds 1−8 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3-OAc 6-OAc 12-OAc 13-OAc

1a 42.3, 90.9, 86.8, 34.4, 29.9, 82.7, 74.8, 48.7, 76.5, 50.9, 73.1, 71.9, 71.0, 42.9, 23.3, 24.2, 50.8, 30.7, 17.7, 23.9, 22.4, 169.9, 21.3, 171.2, 20.9, 170.7, 21.1, 170.1,

2a d

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

41.1, 89.0, 86.9, 32.8, 31.3, 76.7, 76.0, 48.0, 76.1, 49.6, 73.4, 70.5, 71.0, 43.9, 23.3, 23.2, 48.9, 31.2, 18.5, 23.4, 21.0, 169.9,

20.7, 170.5, 22.4, 170.2,

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

CH3 C CH3 C

3b 41.1, 87.3, 86.6, 31.3, 29.2, 80.1, 74.3, 47.9, 75.7, 48.5, 73.6, 70.5, 23.9, 39.9, 23.1, 24.0, 47.8, 30.5, 19.2, 22.5, 22.4, 169.9, 21.2, 170.9, 20.9, 170.4,

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

4a 40.5, 86.1, 86.7, 29.9, 31.4, 74.4, 75.5, 47.2, 75.8, 47.3, 73.9, 69.8, 23.6, 40.6, 23.2, 22.9, 46.6, 30.8, 22.7, 20.3, 22.5, 169.9,

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

44.8, 91.3, 86.6, 35.3, 29.0, 84.7, 75.4, 45.9, 79.9, 51.6, 147.8, 71.2, 30.6, 35.6, 23.0, 23.6, 113.3, 28.6, 15.6, 21.8,

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

44.6, 90.9, 86.2, 34.4, 28.9, 83.1, 75.2, 45.9, 78.9, 50.7, 143.6, 72.8, 75.6, 40.5, 23.2, 23.6, 114.8, 29.8, 16.6, 23.6,

7c

8c

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

44.5, 91.1, 86.3, 34.5, 28.3, 83.4, 75.3, 45.9, 78.7, 50.9, 140.8, 77.2, 71.9, 44.1, 23.2, 23.6, 116.8, 30.1, 16.2, 24.1,

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

21.4, CH3 171.6, C

21.5, 171.8, 21.4, 170.9,

CH3 C CH3 C

20.8, CH3 170.3, C 21.5, CH3 170.4, C

2-phenylacetate

171.9, 42.0, 134.5, 129.2, 128.6, 127.0, 13.7, 18.4, 37.4, 172.3,

3-OCOPr

a

5c

C CH2 C CH CH CH CH3 CH2 CH2 C

13.7, 18.4, 37.4, 172.2,

CH3 CH2 CH2 C

43.4, 88.9, 86.1, 22.5, 30.3, 77.2, 76.1, 45.7, 77.7, 49.7, 143.4, 71.9, 75.8, 41.7, 23.4, 22.7, 112.6, 30.2, 18.0, 23.3,

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

21.5, CH3 170.6, C

13.7, 18.4, 37.4, 172.2,

CH3 CH2 CH2 C

13.6, 18.4, 37.4, 172.3,

CH3 CH2 CH2 C

100 MHz in acetone-d6. b125 MHz in acetone-d6. c100 MHz in CDCl3. dMultiplicities deduced by DEPT.

■

RESULTS AND DISCUSSION The EtOAc-soluble material from the organic extract was separated by silica gel column chromatography to yield 31 fractions as reported earlier.9 The fractions containing terpenoids, as revealed by 1H NMR spectra, were further purified by normal- and reversed-phase HPLC to afford 1−11 (see Experimental Section). The HRESIMS spectrum of klymollin I (1) exhibited a [M + Na]+ peak at m/z 613.2397 [3:1, (M + Na)+/(M + 2 + Na)+] and established a molecular formula of C28H43ClO11Na, requiring seven degrees of unsaturation, and the IR spectrum of 1 revealed the presence of a carbonyl functionality from an absorption at 1734 cm−1. The 13C NMR spectroscopic data of 1 exhibited 28 carbon signals (Table 1), which were assigned by the assistance of a DEPT spectrum to eight methyls, four sp3 methylenes, nine sp3 methines (including five oxymethines), four sp2 carbonyls, and three sp3 oxygenated quaternary carbons. Four ester carbonyls (δC 171.2, 170.7, 170.1, and 169.9) were assigned from the 13C NMR spectrum and were HMBC correlated with four acetate methyls (δH 2.07, 2.04,

1.99, and 1.98 s, respectively). Therefore, the remaining three degrees of unsaturation identified compound 1 as a tricyclic compound. Proton signals (Table 2) resonating at δH 2.49 (1H, m), 2.71 (1H, t, J = 7.2), 3.94 (1H, s), and 4.88 (1H, ddd, 11.6, 7.2, 4.4) and carbon signals appearing at δC 42.3, 50.9, 90.9, and 76.5 indicated the presence of the tetrahydrofuran structural unit of the eunicellins.9,13−16 Also in the 1H NMR spectrum, two doublets at δH 1.08 and 0.97 (each 3H, d, J = 7.2 Hz) arose from two methyls of an isopropyl group. Furthermore, the downfield chemical shifts for H3-15 (δ 1.53) and C-3 (δ 86.8) and the upfield chemical shifts of H3-16 (δ 1.20) and C-7 (δ 74.8) determined the positions of the other acetate and hydroxy group at C-3 and C-7, respectively. The carbon signal of CH2-17, resonating at 50.8 ppm, was more shielded than C11 (δC 73.1) and was HSQC correlated with the methylene proton signals at δH 3.74 and 3.69, suggesting the attachment of a chlorine and an oxygen at C-17 and C-11, respectively.9 The gross structure of metabolite 1 was further confirmed by analysis of COSY and HMBC correlations (Figure 1). From the COSY spectrum of 1, it was possible to identify two structural 1662

dx.doi.org/10.1021/np400372v | J. Nat. Prod. 2013, 76, 1661−1667

Journal of Natural Products

Article

Table 2. 1H NMR Data for Compounds 1−5 1a

position 1 2 4 5 6 8α 8β 9 10 12 13 14 15 16 17 18 19 20 3-OAc 6-OAc 12-OAc 13-OAc 2-phenylacetate

2.49, 3.94, 2.41, 2.11, 1.52, 1.72, 5.68, 1.77, 1.94, 4.88, 2.71, 5.33, 5.17, 1.69, 1.96, 1.53, 1.20, 3.74, 3.69, 1.84, 0.97, 1.08, 2.04, 1.98, 2.07, 1.99,

m s m m m m brs dd (14.4, 4.0)d m ddd (11.6, 7.2, 4.4) t (7.2) d (2.8) dd (8.4, 2.0) m m s s d (12.0) d (12.0) m d (7.2) d (7.2) s s s s

2a 2.61, 4.11, 2.34, 1.96, 1.51, 1.70, 4.50, 1.70, 1.91, 4.78, 2.61, 5.24, 5.32, 1.72, 1.96, 1.58, 1.11, 3.70, 3.61, 1.80, 1.03, 1.08, 2.02,

3b

m s m m m m brs m m brd (11.6) m d (2.8) brs m m s s d (11.6) d (11.2) m d (6.8) d (6.8) s

2.06, s 1.98, s

3-OCOPr

4a

5c

2.66, 3.96, 2.30, 2.19, 1.58,

m d (6.5) t (13.0) m m

2.73, 3.97, 2.16, 2.08, 1.56,

t (8.0) d (8.0) m m m

2.25, 3.71, 2.55, 2.06, 1.46,

m brs dd (15.2, 8.8) m m

5.71, 1.60, 1.95, 4.71, 2.51, 5.11, 2.01, 1.39, 1.65, 1.67, 1.22, 3.72, 3.62, 1.79, 1.03, 1.03, 1.97, 1.98, 2.04,

d (8.5) m m brd (12.0) m dd (11.0, 3.5) m m m s s d (11.0) d (11.0) m d (6.5) d (6.5) s s s

4.45, 1.58, 1.89, 4.64, 2.43, 5.08, 2.06,

brs m m dd (12.4, 3.6) d (7.6) dd (11.6, 3.2) m

5.60, d (5.6) 1.80, m

1.71, 1.65, 1.11, 3.70, 3.56, 1.74, 1.04, 1.07, 1.94,

m s s d (11.2) d (11.2) m d (6.4) d (6.4) s

4.45, 2.95, 4.40, 1.87,

dd (14.4, 7.2) t (7.2) brs m

1.84, 1.39, 1.10, 4.82, 5.00, 1.82, 0.79, 0.98,

m s s brs brs m d (7.2) d (7.2)

3.63, 7.27, 7.31, 7.25, 0.98, 1.66, 2.28,

s m m m t (7.2) m m

2.06, s

a Spectra recorded at 400 MHz in acetone-d6 at 25 °C. bSpectra recorded at 500 MHz in acetone-d6 at 25 °C. cSpectra recorded at 400 MHz in CDCl3 at 25 °C. dJ values in Hz in parentheses.

Figure 1. COSY and HMBC correlations for 1, 5, and 7.

and between H3-15 and both H-2 and H-6 suggested that H-2, H-6, H-9, H-14, and H3-15 are all α-oriented. Thus, the structure of diterpenoid 1 was established. The HRESIMS spectrum of 2 exhibited a pseudomolecular ion peak at m/z 571.2281 [3:1, (M + Na)+/(M + 2 + Na)+] consistent with a molecular formula of C26H41ClO10 and implying six degrees of unsaturation. By comparison of the NMR data of 2 with those of 1 (Tables 1 and 2), it was found that an acetate group in 1 attached at C-6 was replaced by a hydroxy group in 2, as confirmed by downfield shifted peaks (δC 76.7 and δH 4.50) relative to those of 1 (δC 82.7 and δH 5.68). Further analysis of the NOE interactions revealed that 2 possessed the same relative configurations at C-1, C-2, C-3, C6, C-9, C-10, C-11, C-12, C-13, and C-14 as those of 1.

units, which were further assembled by key HMBC correlations from H-2 to C-1, C-9, and C-10; H3-15 to C-2, C-3, and C-4; H2-4 to C-6; H3-16 to C-6, C-7, and C-8; H2-17 to C-10, C-11, and C-12; and both H3-19 and H3-20 to C-14 and C-18. Moreover, the HMBC correlations of H-6, H-12, and H-13 to three acetate carbonyl carbons, respectively, showed the presence of three acetoxy groups at C-6, C-12, and C-13. In the NOESY spectrum of 1 (Figure 2), NOE correlations between H-10 and H2-17 (δ 3.69), H-1, and one proton (δH 1.94) at C-8, which was assigned as H-8β (δH 1.94); H3-16 and H2-8 (δ 1.94 and 1.77); H2-17 (δ 3.74) and both H-10 and H13; and H-1 and both H2-17 (δ 3.69) and H-12, suggested that H-1, H-10, H-12, H-13, H3-16, and H2-17 are all β-oriented. Also, NOE interactions between H-14 and both H-2 and H-9 1663

dx.doi.org/10.1021/np400372v | J. Nat. Prod. 2013, 76, 1661−1667

Journal of Natural Products

Article

Figure 2. Key NOESY correlations of 1.

Table 3. 13C NMR Data for Compounds 9 and 10

Klymollin K (3) was obtained as a colorless oil that gave a pseudomolecular ion peak at m/z 555.2341 [3:1, (M + Na)+/ (M + 2 + Na)+] by HRESIMS, appropriate for a molecular formula of C 26 H 41 ClO 9 and implying six degrees of unsaturation. By means of extensive 2D NMR experiments (COSY, HSQC, and HMBC), the structure of 3 was found to be close to that of 1 except that the ester group at C-13 in 1 was replaced by a hydrogen atom. A structurally related metabolite, klymollin L (4), was also isolated as a colorless oil with a molecular formula of C24H39ClO8, implying five degrees of unsaturation. NMR spectroscopic data of 4 showed the presence of two acetoxy groups. Comparison of the NMR data of 4 with those of 2 revealed that the only difference between the two compounds arises from the replacement of the acetoxy moiety at C-13 in 2 by a hydrogen atom in 4. The HRESIMS spectrum of 5 exhibited a pseudomolecular ion peak at m/z 565.3138 [M + Na]+, consistent with a molecular formula of C32H46O7 and implying 10 degrees of unsaturation. The IR spectrum of 5 revealed the presence of hydroxy and carbonyl functionalities from absorptions at 3439 and 1732 cm−1, respectively. An ester carbonyl (δC 171.9) and phenyl carbons (δC 134.5, 129.2, 128.6, and 127.0) were assigned from the 13C NMR spectrum and were correlated by an HMBC spectrum with the methylene protons (δH 3.63 s, 2H) of a 2-phenylacetate. Further, the other ester carbonyl (δC 172.3) was HMBC correlated with the methylene protons (δH 2.28 m, 2H and 1.66, m, 2H) of one n-butyrate. By comparison of the NMR data of 5 with those of litophynin I,17 it was found that a hydroxy group attached at C-6 of litophynin I was replaced by a phenylacetoxy group in 5. This was further evidenced by the HMBC correlations observed from H3-16 (δ 1.10, 3H, s) to C-6 (δ 84.7, CH), C-7 (δ 75.4, C), and C-8 (δ 45.9, CH2) and from H-6 (δ 5.60) to the 2-phenylacetate resonating at δC 171.9 (C). Thus, the structure of klymollin M (5) was established and was found to be the first eunicellinbased metabolite bearing a phenylacetate. Klymollin N (6) was obtained as a colorless oil that gave a pseudomolecular ion peak at m/z 547.2880 [M + Na]+ in the HRESIMS spectrum, consistent with the molecular formula C28H44O9 and implying seven degrees of unsaturation. By comparison of the NMR data of 6 with those of 5 (Tables 1, 2, and 4), it was found that a 2-phenylacetate at C-6 in 5 was replaced by an acetoxy group in 6. Moreover, the hydrogen atom attached at C-13 of 5 was replaced by an acetoxy group in

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

9a 44.8, 91.2, 86.8, 35.2, 29.2, 84.0, 75.4, 46.2, 79.9, 51.5, 147.8, 71.2, 30.6, 35.6, 23.0, 23.6, 113.3, 28.6, 15.6, 21.8, 22.4, 169.5, 21.6, 171.8,

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

10a 44.3, 90.7, 86.7, 35.0, 30.4, 79.1, 76.7, 46.1, 79.5, 51.1, 148.0, 70.8, 31.0, 36.4, 23.3, 22.5, 112.0, 29.0, 16.3, 21.9, 22.4, 169.5,

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

100 MHz in CDCl3. bMultiplicities deduced by DEPT.

6. This was further evidenced by the HMBC correlations observed from H-6 (δ 5.64) and H-13 (δ 4.91) to the carbonyl carbons resonating at δC 171.6 (C) and 170.4, respectively. Klymollin O (7), with a molecular formula of C28H44O9, was obtained as a colorless oil. Careful comparison of its 1H and 13C NMR data with those of 6 suggested that 7 has the same molecular formula and showed that a hydroxy group at C-12 and the acetoxy group at C-13 in 6 were replaced by an acetoxy and a hydroxy group in 7, respectively, as confirmed by the downfield shifted δC value of C-12 (δC 72.8) of 6 relative to that of 7 (δC 77.2) and the HMBC correlation from H-12 (δ 5.44) to the carbonyl carbon resonating at δ 170.9. A structurally similar metabolite, klymollin P (8), was also isolated as a colorless oil with a molecular formula of C26H42O8, implying six degrees of unsaturation. Comparison 1664

dx.doi.org/10.1021/np400372v | J. Nat. Prod. 2013, 76, 1661−1667

Journal of Natural Products

Article

Table 4. 1H NMR Data for Compounds 6−10 6a

7a

8a

1

2.27, m

2.24, m

2

3.79, s

3.79, s

position

4

5 6 8 9

10

2.51, dd (9.6, 4.8)b 2.06, m 1.47, m 1.64, m 5.64, d (6.0) 1.83, m 4.66, dd (14.0, 7.2) 2.90, t (6.8)

12

4.35, d (2.4)

13

4.91, dd (10.0, 2.8)

14 15 16 17 18 19 20 3-nbutyrate

3-OAc 6-OAc 11-OAc 12-OAc 13-OAc

2.14, brs 1.47, s 1.20, s 5.01, s 5.16, s 1.76, m 0.91, d (7.2) 1.08, d (7.2) 0.98, t (7.2)

9a

10a

2.38, m

2.23, t (9.6) 3.73, s

2.07, m

3.88, d (4.0) 2.00, m

1.42, m 1.55, m 5.64, brs

1.48, m 1.81, m 4.55, brs

1.81, m

1.79, m

2.56, dd (14.4, 8.4) 2.05, m 1.48, m 1.56, m 5.66, d (6.4) 1.82, m

2.28, td (7.2, 2.0) 3.67, d (2.0) 2.50, dd (9.6, 5.2)

4.44, m

4.71, m

4.50, m

4.51, m

2.92, t (6.4) 5.44, d (2.8)

2.77, dd (3.6, 3.2) 4.33, brs

2.95, t (6.8)

3.74, dd (6.8, 3.2)

5.02, dd (7.6, 5.2)

1.82, m

2.87, t (6.4) 4.37, d (2.8) 1.84, m 1.40, m

1.63, m 2.27, m

1.80, m 1.47, s 1.19, s 5.07, s 5.21, s 2.18, m 0.97, d (7.2) 1.16, d (7.2) 0.99, t (6.8) 1.60, m 2.20, m

2.07, s

2.14, s

2.08, m 1.55, s 1.17, s 5.11, s 5.21, s 1.70, m 0.98, d (6.8) 1.08, d (6.8) 0.96, t (7.2) 1.63, m 2.22, m

4.41, d (4.0)

1.32, m 1.86, m 1.43, s 1.20, s 4.83, s 5.02, s 1.81, m 0.81, d (7.2) 0.99, d (7.2)

unsaturation. By comparing the NMR data of 11 with those of 10 (Tables 3−5), it was found that a hydroxy-bearing Table 5. 1H and 13C NMR Data for Compound 11 position

1.90, m 1.68, m 4.53, d (7.6) 1.83, m

1.82, m 1.46, s 1.16, s 4.86, s 5.04, s 1.78, m 0.83, d (6.8) 0.99, d (6.8)

43.6,a CHb 90.1, CH 84.9, C 28.4, CH2

5

35.6, CH2

6 7 8

72.2, CH 150.9, C 38.5, CH2

9 10 11 12

81.9, 45.7, 146.5, 71.7,

CH CH C CH

13

31.0, CH2

14 15

35.8, CH 21.9, CH3

16

116.6, CH2

17

116.1, CH2

18 19 20 3-OAc

26.9, 15.4, 21.7, 22.6, 169.9,

CH CH3 CH3 CH3 C

δH 2.24,c m 3.79, s 2.25, 1.71, 1.70, 2.17, 4.40,

m m m m brs

2.27, 2.78, 4.53, 3.07,

m dd (14.0, 4.8)d dd (10.4, 4.4) t (10.0)

4.44, brs 1.87, 1.32, 1.86, 1.64,

m d (10.8) m s

5.11, 5.46, 4.86, 5.11, 1.98, 0.77, 0.99, 1.93,

s s s s m d (6.8) d (6.8) s

a

2.10, s 2.07, s

100 MHz in CDCl3. bMultiplicities deduced by DEPT. cSpectra recorded at 400 MHz in CDCl3 at 25 °C. dJ values in Hz in parentheses.

2.07, s

2.08, s 2.11, s

δC

1 2 3 4

quaternary carbon connected to a methyl in 10 was replaced by a 7,16-double bond in 11. This was further evidenced by the HMBC correlations observed from H2-16 (2H, δ 5.11, s and δ 5.46, s) to C-6 (δ 72.2, CH), C-7 (δ 150.9, C), and C-8 (δ 38.5, CH2). The absolute configuration of klymollin C9 has been completely assigned based on NOE correlations and Mosher’s method. Compounds 1−11 are likely to be in the same enantiomeric series as klymollin C based on a shared biosynthetic pathway. Thus, these compounds are suggested to possess the absolute configurations as shown in formulas 1− 11. Although many eunicellin-type natural products have been discovered, a eunicellin containing a 2-phenylacetate (5) was discovered for the first time. The cytotoxicities of metabolites 1−11 against a limited panel of human tumor cell lines including human erythro myeloblastoid leukemia (K562), human acute lymphoblastic leukemia (Molt-4), and human breast carcinoma (T47D) were investigated. Compound 5 showed cytotoxicity toward K562, Molt-4, and T47D cancer cell lines with ED50 values 7.97 ± 2.55, 4.35 ± 0.63, and 8.58 ± 1.72 μM, respectively. The above results suggest that eunicellin-

2.10, s

a Spectra recorded at 400 MHz in CDCl3 at 25 °C. bJ values in Hz in parentheses.

of the NMR data of 8 with those of 6 revealed that the only difference between the two compounds arises from the replacement of the acetoxy moiety at C-6 in 6 by a hydroxy group in 8, as confirmed by the upfield shifted δC value of C-6 (δC 83.1) of 6 relative to that of 8 (δC 77.2). Klymollins Q (9) and R (10) were also isolated as colorless oils. The molecular formulas of 9 and 10 determined by HRESIMS were C24H38O7 and C22H36O6, respectively. Except for the absence of the phenylacetate at C-6, the 1H and 13C NMR spectroscopic data of 9 and 10 were found to be very close to those of 5, indicating very similar structures for these three metabolites. By comparison of the NMR data of 9 with those of 10 (Tables 3 and 4), it was found that the acetoxy group at C-6 in 9 was replaced by a hydroxy group in 10. The HRESIMS spectrum of 11 exhibited a pseudomolecular ion peak at m/z 401.2302 [M + Na]+, consistent with a molecular formula of C22H34O5 and implying six degrees of 1665

dx.doi.org/10.1021/np400372v | J. Nat. Prod. 2013, 76, 1661−1667

Journal of Natural Products

Article

Table 6. Inhibitory Effects of Compounds 1−11 on Superoxide Anion Generation and Elastase Release by Human Neutrophilsa superoxide anion compound

IC50 (μM)b

1 2 3 4 5 6 7 8 9 10 11

>10 >10 >10 >10 3.13 ± 0.39 >10 >10 >10 >10 >10 >10

elastase release IC50 (μM)b

inh % 9.36 5.02 8.92 3.37 81.56 10.46 2.92 1.17 14.74 7.14 3.20

± ± ± ± ± ± ± ± ± ± ±

2.17 1.82 3.19 1.14 3.23 3.52 1.16 1.33 4.32 3.09 0.67

* * * *** *

* **

>10 >10 >10 >10 2.92 ± 0.27 >10 >10 >10 >10 >10 >10

inh % 6.42 7.30 −1.19 9.18 89.16 6.47 16.75 7.35 7.33 11.55 0.62

± ± ± ± ± ± ± ± ± ± ±

1.37 3.74 2.49 3.92 5.77 2.06 2.08 3.32 4.34 4.14 2.35

**

*** * **

*

Percentage of inhibition (inh %) at 10 μM concentration. Results are presented as mean ± SEM (n = 3 or 4). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control value. bConcentration necessary for 50% inhibition (IC50). a

Klymollin I (1): yellow oil; [α]23D = +53 (c 1.91, CHCl3); IR (neat) νmax 3460 and 1734 cm−1; 13C and 1H NMR data (400 MHz; acetoned6), see Tables 1 and 3; ESIMS m/z 613 [M + Na]+; HRESIMS m/z 613.2397 [M + Na]+ (calcd for C28H43ClO11Na, 613.2391). Klymollin J (2): yellow oil; [α]23D = +65 (c 1.51, CHCl3); IR (neat) νmax 3458 and 1735 cm−1; 13C and 1H NMR data (400 MHz; acetoned6), see Tables 1 and 3; ESIMS m/z 571 [M + Na]+; HRESIMS m/z 571.2281 [M + Na]+ (calcd for C26H41ClO10Na, 571.2286). Klymollin K (3): colorless oil; [α]23D = +86 (c 0.94, CHCl3); IR (neat) νmax 3479 and 1732 cm−1; 13C and 1H NMR data (500 MHz; acetone-d6), see Tables 1 and 3; ESIMS m/z 555 [M + Na]+; HRESIMS m/z 555.2341 [M + Na]+ (calcd for C26H41ClO9Na, 555.2337). Klymollin L (4): colorless oil; [α]23D = +47 (c 1.02, CHCl3); IR (neat) νmax 3446 and 1733 cm−1; 13C and 1H NMR data (400 MHz; acetone-d6), see Tables 1 and 3; ESIMS m/z 513 [M + Na]+; HRESIMS m/z 513.2234 [M + Na]+ (calcd for C24H39ClO8Na, 513.2231). Klymollin M (5): colorless oil; [α]23D = +71 (c 0.97, CHCl3); IR (neat) νmax 3439, 1732, and 1603 cm−1; 13C and 1H NMR data (400 MHz; CDCl3), see Tables 1 and 3; ESIMS m/z 565 [M + Na]+; HRESIMS m/z 565.3138 [M + Na]+ (calcd for C32H46O7Na, 565.3141). Klymollin N (6): colorless oil; [α]23D = +40 (c 0.80, CHCl3); IR (neat) νmax 3445 and 1731 cm−1; 13C and 1H NMR data (400 MHz; CDCl3), see Tables 1 and 4; ESIMS m/z 547 [M + Na]+; HRESIMS m/z 547.2880 [M + Na]+ (calcd for C28H44O9Na, 547.2883). Klymollin O (7): colorless oil; [α]23D = +30 (c 0.34, CHCl3); IR (neat) νmax 3445 and 1732 cm−1; 13C and 1H NMR data (400 MHz; CDCl3), see Tables 1 and 4; ESIMS m/z 547 [M + Na]+; HRESIMS m/z 547.2880 [M + Na]+ (calcd for C28H44O9Na, 547.2883). Klymollin P (8): colorless oil; [α]23D = +38 (c 0.80, CHCl3); IR (neat) νmax 3445 and 1732 cm−1; 13C and 1H NMR data (400 MHz; CDCl3), see Tables 1 and 4; ESIMS m/z 505 [M + Na]+; HRESIMS m/z 505.2774 [M + Na]+ (calcd for C26H42O8Na, 505.2777). Klymollin Q (9): yellow oil; [α]23D = +30 (c 1.08, CHCl3); IR (neat) νmax 3445 and 1732 cm−1; 13C and 1H NMR data (400 MHz; CDCl3), see Tables 2 and 4; ESIMS m/z 461 [M + Na]+; HRESIMS m/z 461.2513 [M + Na]+ (calcd for C24H38O7Na, 461.2515). Klymollin R (10): colorless oil; [α]24D = +19 (c 1.51, CHCl3); IR (neat) νmax 3420 and 1733 cm−1; 13C and 1H NMR data (400 MHz; CDCl3), see Tables 2 and 4; ESIMS m/z 419 [M + Na]+; HRESIMS m/z 419.2411 [M + Na]+ (calcd for C22H36O6Na, 419.2409). Klymollin S (11): white solid; mp 141−143 °C; [α]23D = +123 (c 0.22, CHCl3); IR (neat) νmax 3443 and 1732 cm−1; 13C and 1H NMR data (400 MHz; CDCl3), see Tables 2 and 5; ESIMS m/z 401 [M + Na]+; HRESIMS m/z 401.2304 [M + Na]+ (calcd for C22H34O5Na, 401.2302). Cytotoxicity Testing. Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays of

based metabolites bearing a phenylacetate as in 5 may have enhanced cytotoxicity. It is known that the toxic reactive oxygen species and proteolysis enzymes produced by neutrophils can destroy the surrounding tissue and have a central role in the pathogenesis of various inflammatory diseases.24,25 The effects on neutrophil pro-inflammatory responses of compounds 1−11 were evaluated by suppressing N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion (O2•−) generation and elastase release in human neutrophils, as shown in Table 6. From the results, 5 showed strong inhibition (81.56 ± 3.23%) toward superoxide anion generation. Klymollin M (5) also exhibited potent inhibitory activity against elastase release, with 89.16 ± 5.77% inhibition in the same fMLP/CB-stimulated cells. Compound 5 was found to be the most potent in inhibiting the superoxide generation (IC50 3.13 ± 0.39 μM) and elastase release (IC50 2.92 ± 0.27 μM) in this assay. The ability of 5 to inhibit both superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils might also arise from the presence of the phenylacetate at C-6.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Same as previously reported.9 Animal Material. Klyxum molle was collected in June 2008, as previously described.9 Extraction and Isolation. The EtOAc-soluble material (22 g) from the organic extract was subjected to silica gel column chromatography as reported earlier9 to yield 31 fractions. Fractions 24 and 25, eluting with EtOAc (100%), were rechromatographed over a reversed-phase RP-18 column using MeOH and H2O (8:1) as the mobile phase to afford four subfractions (A1−A4). Subfractions A2 and A3 were separated by reversed-phase HPLC (CH3CN−H2O, 1:2.3) to afford compounds 1 (6.7 mg), 2 (5.3 mg), 3 (3.3 mg), 5 (3.4 mg), and 11 (2.8 mg), respectively. Fraction 26, eluted with EtOAc− acetone (1:1), was rechromatographed over a reversed-phase RP-18 column using MeOH and H2O (8:1) as the mobile phase to afford four subfractions (B1−B4). Subfractions B2 and B3 were separated by reversed-phase HPLC (CH3CN−H2O, 1:2.2) to afford compounds 6 (2.8 mg), 7 (1.2 mg), and 9 (5.3 mg), respectively. Fraction 29, eluted with acetone−MeOH (1:1), was rechromatographed over a reversedphase RP-18 column using MeOH and H2O (8:1) as the mobile phase to afford four subfractions (C1−C4). Subfractions C2 and C3 were separated by reversed-phase HPLC (CH3CN−H2O, 1:1.6) to afford compounds 4 (3.6 mg), 8 (3.8 mg), and 10 (0.8 mg), respectively. 1666

dx.doi.org/10.1021/np400372v | J. Nat. Prod. 2013, 76, 1661−1667

Journal of Natural Products

Article

compounds 1−11 were performed using the Alamar Blue assay.18,19 5Fluorouracil, employed as positive control, exhibited cytotoxic activity toward K562, Molt-4, and T47D cancer cell lines with ED50 values of 16.22 ± 1.77, 15.07 ± 1.61, and 50.20 ± 13.22 μM, respectively. Preparation of Human Neutrophils. Human neutrophils obtained from peripheral blood of healthy adult volunteers (20−30 years old) were enriched using a standard method of dextran sedimentation, Ficoll Hypaque centrifugation, and hypotonic lysis.20,21 Purified neutrophils were resuspended in a Ca2+-free HBSS buffer (pH 7.4) at 4 °C prior to use. Measurement of O2•− Generation. The O2•− production was assayed by the method based on the superoxide oxide dismutaseinhibitable reduction of ferricytochrome c.22−24 Briefly, neutrophils (6 × 105/mL) incubated with ferricytochrome c (0.5 mg/mL) and Ca2+ (1 mM) were equilibrated at 37 °C for 2 min and then treated with DMSO (as control) or different concentrations of compounds for 5 min. Neutrophils were activated by 100 nM fMLP for 10 min in the pretreatment of cytochalasin B (CB, 1 μg/mL) for 3 min (fMLP/CB). Measurement of Elastase Release. The elastase release was assayed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the enzyme substrate.23,24 Briefly, neutrophils (6 × 105/mL) incubated with MeOSuc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM) were equilibrated at 37 °C for 2 min and treated with compounds for 5 min. Neutrophils were then activated with fMLP (100 nM)/CB (0.5 μg/mL) for 10 min. Statistical Analysis. Results are expressed as the mean ± SEM, and comparisons were made using Student’s t-test. A probability of 0.05 or less was considered significant. The software SigmaPlot was used for the statistical analysis.

■

(8) Chen, B.-W.; Huang, C.-Y.; Wen, Z.-H.; Su, J.-H.; Wang, W.-H.; Sung, P.-J.; Wu, Y.-C.; Sheu, J.-H. Bull. Chem. Soc. Jpn. 2011, 84, 1237−1242. (9) Hsu, F.-J.; Chen, B.-W.; Wen, Z.-H.; Huang, C.-Y.; Dai, C.-F.; Su, J.-H.; Wu, Y.-C.; Sheu, J.-H. J. Nat. Prod. 2011, 74, 2467−2471. (10) Ahmed, A. F.; Wu, M.-H.; Wang, G.-H.; Wu, Y.-C.; Sheu, J.-H. J. Nat. Prod. 2005, 68, 1051−1055. (11) Williams, D. E.; Amlani, A.; Dewi, A. S.; Patrick, B. O.; van Ofwegen, L.; Mui, A. L.-F.; Andersen, R. J. Aust. J. Chem. 2010, 63, 895−900. (12) Bowden, B. F.; Coll, J. C.; Dai, M. C. Aust. J. Chem. 1989, 42, 665−673. (13) Hassan, H. M.; Khanfar, M. A.; Elnagar, A. Y.; Mohammed, R.; Shaala, L. A.; Youssef, D. T. A.; Hifnawy, M. S.; El Sayed, K. A. J. Nat. Prod. 2010, 73, 848−853. (14) Ciavatta, M. L.; Manzo, E.; Mollo, E.; Mattia, C. A.; Tedesco, C.; Irace, C.; Guo, Y.-W.; Li, X.-B.; Cimino, G.; Gavagnin, M. J. Nat. Prod. 2011, 74, 1902−1907. (15) Chen, Y.-H.; Tai, C.-Y.; Kuo, Y.-H.; Kao, C.-Y.; Li, J.-J.; Hwang, T.-L.; Fang, L.-S.; Wang, W.-H.; Sheu, J.-H.; Sung, P.-J. Chem. Pharm. Bull. 2011, 59, 353−358. (16) Chen, Y.-H.; Tai, C.-Y.; Hwang, T.-L.; Weng, C.-F.; Li, J.-J.; Fang, L.-S.; Wang, W.-H.; Wu, Y.-C.; Sung, P.-Y. Mar. Drugs 2010, 8, 2936−2945. (17) Ochi, M.; Yamada, K.; Kataoka, K.; Kotsuki, H.; Shibata, K. Chem. Lett. 1992, 155−158. (18) Nakayama, G. R.; Caton, M. C.; Nova, M. P.; Parandoosh, Z. J. Immunol. Methods 1997, 204, 205−208. (19) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421−5426. (20) Boyum, A. Scand. J. Clin. Lab. Invest. 1968, 97, 77−89. (21) Jauregui, H. O.; Hayner, N. T.; Driscoll, J. L.; Williams-Holland, R.; Lipsky, M. H.; Galletti, P. M. In Vitro 1981, 17, 1100−1110. (22) Babior, B. M.; Kipnes, R. S.; Curnutte, J. T. J. Clin. Invest. 1973, 52, 741−744. (23) Hwang, T. L.; Leu, Y. L.; Kao, S. H.; Tang, M. C.; Chang, H. L. Free Radical Biol. Med. 2006, 41, 1433−1441. (24) Yang, S.-C.; Chung, P.-J.; Ho, C.-M.; Kuo, C.-Y.; Hung, M.-F.; Huang, Y.-T.; Chang, W.-Y.; Chang, Y.-W.; Chan, K.-H.; Hwang, T.-L. J. Immunol. 2013, 190, 6511−6519. (25) Mantovani, A.; Cassatella, M. A.; Costantini, C.; Jaillon, S. Nat. Rev. Immunol. 2011, 11, 519−531.

ASSOCIATED CONTENT

S Supporting Information *

1 H and 13C NMR spectra for 1−11. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +886-7-5252000, ext. 5030. Fax: +886-7-5255020. Email: [email protected]. Author Contributions ▽

M.-C. Lin and B.-W. Chen provided equal contributions to this work. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Financial support awarded to J.-H.S was provided by the National Science Council of Taiwan (NSC-100-2320-B-110001-MY2) and Aim for the Top University Program from the Ministry of Education of Taiwan (01C030205).

■

REFERENCES

(1) Chill, L.; Berrer, N.; Benayahu, Y.; Kashman, Y. J. Nat. Prod. 2005, 68, 19−25. (2) Chen, B.-W.; Wu, Y.-C.; Chiang, M. Y.; Su, J.-H.; Wang, W.-H.; Fan, T.-Y.; Sheu, J.-H. Tetrahedron 2009, 65, 7016−7022. (3) Chen, B.-W.; Chao, C.-H.; Su, J.-H.; Tsai, C.-W.; Wang, W.-H.; Wen, Z.-H.; Huang, C.-Y.; Sung, P.-J.; Wu, Y.-C.; Sheu, J.-H. Org. Biomol. Chem. 2011, 9, 834−844. (4) Chen, B.-W.; Su, J.-H.; Wen, Z.-H.; Sung, P.-J.; Sheu, J.-H. Org. Biomol. Chem. 2010, 8, 2363−2366. (5) Wu, S.-L.; Su, J.-H.; Wen, Z.-H.; Hsu, C.-H.; Chen, B.-W.; Dai, C.-F.; Kuo, Y.-H.; Sheu, J.-H. J. Nat. Prod. 2009, 72, 994−1000. (6) Wu, S.-L.; Su, J.-H.; Lu, Y.; Chen, B.-W.; Huang, C.-Y.; Wen, Z.H.; Kuo, Y.-H.; Sheu, J.-H. Bull. Chem. Soc. Jpn. 2011, 84, 626−632. (7) Tai, C.-J.; Su, J.-H.; Huang, M.-S.; Wen, Z.-H.; Dai, C.-F.; Sheu, J.-H. Mar. Drugs 2011, 9, 2036−2045. 1667

dx.doi.org/10.1021/np400372v | J. Nat. Prod. 2013, 76, 1661−1667