Cytotoxic Polyketide Derivatives from the South China Sea Sponge

Mar 25, 2013 - the South China Sea sponge Plakortis simplex. Their structures were identified by spectroscopic and chemical methods, including NMR, MS...
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Cytotoxic Polyketide Derivatives from the South China Sea Sponge Plakortis simplex Jinrong Zhang,†,∥ Xuli Tang,‡,∥ Jing Li,† Peifeng Li,‡ Nicole J. de Voogd,§ Xiaoqin Ni,† Xiaojie Jin,⊥ Xiaojun Yao,⊥ Pinglin Li,*,† and Guoqiang Li*,† †

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, People’s Republic of China § National Museum of Natural History, PO Box 9517, 2300 RA Leiden, The Netherlands ⊥ State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, People’s Republic of China S Supporting Information *

ABSTRACT: Five new polyketides, plakortoxides A (1) and B (2), simplextones C (3) and D (4), and plakorsin D (5), together with six known analogues (6−11) were isolated from the South China Sea sponge Plakortis simplex. Their structures were identified by spectroscopic and chemical methods, including NMR, MS, and IR. Experimental and calculated ECD spectra and the modified Mosher’s method were used to determine the absolute configurations. Structurally, both plakortoxides A and B feature a butenolide coupled to an epoxide moiety, while simplextones C and D consist of γbutyrolactone and cyclopentane moieties, and plakorsin D is a furan acetic acid polyketide. The cytotoxic activities of the isolates were tested, and compounds 8, 10, and 11 showed potent cytotoxicity against both K562 and HeLa tumor cell lines with IC50 values ranging from 0.8 to 5.3 μM. Compound 3 showed significant inhibitory activity against c-Met kinase.

M

Island in the South China Sea in 2009. Five new polyketides, namely, plakortoxides A (1) and B (2), simplextones C (3) and D (4), and plakorsin D (5), together with six known analogues, woodylides A (6) and B (7),13 simplexolides B (8) and E (9),9 2-[3,5-diethyl-5-(2-ethylhexyl)-2(5H)-furanylidene]acetic acid methyl ester (10),14 and 2-[3,5-diethyl-5-(2-ethyl-3-hexen-1yl)-2(5H)-furanylidene]acetic acid methyl ester (11),15 were isolated from the MeOH extracts. The structures of the new compounds were elucidated by NMR, MS, the modified Mosher’s method, and CD spectroscopy. Structurally, compounds 1 and 2 feature a butenolide coupled to an epoxide moiety, compounds 3 and 4 are analogues of simplextones A and B,12 and compound 5 is a furan acetic acid polyketide. All 11 compounds were assayed for antitumor activity against selected tumor cell lines and for inhibition of c-Met kinase. In this paper, we describe the isolation, structure elucidation, and bioactivities of compounds 1−11.

arine sponges of the cosmopolitan genus Plakortis (Homosclerophorida, Plakinidae), with about 15 species described worldwide, have been fascinating since the 1980s because of the variety of unusual metabolites they generate.1 Continuing studies on the species Plakortis simplex, one of the most prolific species of this genus, have resulted in the discovery of dozens of structurally unique and biologically active metabolites, including cyclic peroxides with 1,2dioxane2−5 or 1,2-dioxolane6 ring systems, furano esters,7 bicyclic lactones,8 and linear polyketides.9 The majority of these polyketides showed interesting biological activities such as cytotoxic and antimalarial properties,3,4 as well as the activation of SR Ca2+-ATPase.8 Extensive studies of Caribbean specimens of P. simplex were initiated by Ernesto Fattorusso’s group in 1997.1,4,10 Ya-Ching Shen’s and Walter Michaelis’s groups isolated structurally unique polyketide analogues from the same species collected near Taiwan in 2001 and from the Sula Ridge off the Norwegian coast in 2005, respectively.5,11 Recently, the Houwen Lin group reported the surprising discovery of the simplextones with an unprecedented polyketide skeleton from specimens collected from the South China Sea in 2011.12 With the aim of searching for new antitumor compounds, we systematically restudied P. simplex collected off YongXing © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Plakortoxide A (1) was obtained as a colorless oil. The molecular formula of 1 was established as C17H28O3 by Received: November 4, 2012 Published: March 25, 2013 600

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triplet methyls observed in the 1H NMR spectrum further indicated that three of the four methyls were coupled to methylene protons (two as ethyl groups). A C6 linear aliphatic chain with a methyl-branch at C-8 was established on the basis of careful analysis of the COSY spectrum guided by HMQC data, as well as the HMBC correlations from H3-17 to C-7, C-8, and C-9 and from H2-9 and H3-12 to C-10. The partial structures mentioned above were connected by interpretation of the HMBC data (Figure 1). Two ethyl groups located at C-4 and C-6, respectively, were deduced from the HMBC correlations between H3-14 and C-4 and between H3-16 and C-6. The crucial cross-peaks of H-5 with C-3, C-4, C-6, and C-7 in the HMBC spectrum clearly indicated that the trisubstituted epoxide moiety was connected to the butenolide with the C7 aliphatic chain. The relative configuration of 1 was determined by NOESY experiments. The NOE correlations (Figure 2) between H-5 and H2-7 and the absence of any correlations between H-5 and H2-15 or H3-16 indicated that both H-5 and H2-7 were oriented on the same side of the epoxide ring, while the ethyl group (H215/H3-16) and the butenolide moieties were oriented on the opposite side. Thus, there were four possible candidate stereostructures (4S/5R/6S, 4S/5S/6R, 4R/5R/6S, and 4R/ 5S/6R) that could be assigned to 1. The absolute configuration of 1 was determined by CD spectroscopy combined with the NOESY experiments. The ECD curves for the four possible candidate stereoisomers were calculated using the TD-DFT theory method at the B3LYP/DGDZVP level (Supporting Information Figures SC1 and SC2).18,19 Visual inspection suggested that either the 4S/5R/6S or 4S/5S/6R curve is approximately the same as the experimental curve with a pronounced negative Cotton effect (CE) at 207 nm (Figure 3). In all stable conformers for the 4S/5R/6S isomer generated from a conformational search by molecular mechanics

HRESIMS and required four degrees of unsaturation. The IR spectrum of 1 indicated the presence of a butenolide (absorption bands at 1759 and 1696 cm−1). The relatively downfield olefinic proton signals at δH 7.80 (1H, d, J = 5.5 Hz, H-3) and 6.29 (1H, d, J = 5.5 Hz, H-2), carbon signals at δC 172.2 (C, C-1), 159.2 (CH, C-3), and 121.1 (CH, C-2), and downfield oxygenated quaternary carbon signal at δC 88.1 (C, C-4) in its 1H and 13C NMR spectra supported this speculation.16,17 Thus, the remaining degree of unsaturation was assigned to a trisubstituted epoxide due to the upfield oxygenated methine proton resonance at δH 3.04 (1H, s, H-5) and carbon resonances at δC 64.5 (CH, C-5) and 65.0 (C, C-6). The additional signals in the 13C NMR spectrum included four methyls, six methylenes, and one methine (Table 2). Three

Table 1. 1H NMR Data for Compounds 1−5 (recorded in DMSO-d6)a 1

2

H

δH

2

6.29,

d (5.5)

6.28,

d (5.5)

3 5 7

7.80, 3.04, 1.85, 0.84, 1.53, 1.25, 1.11, 1.25,

d (5.5) s m m m m m m

7.81, 3.01, 1.60, 1.18, 1.39, 1.22,

1.25, 0.86, 1.96, 1.89, 0.75, 1.79, 1.70, 0.91, 0.88,

m t (6.6) dq (14.3, 7.7) m t (7.7) dq (14.3, 7.7) dq (14.3, 7.7) t (7.7) d (6.6)

8 9 10 11 12 13 14 15 16 17 18

(J in Hz)

δH

4b

3

5c

δH

(J in Hz)

δH

(J in Hz)

δH

dd (18.7, 7.7) dd (18.7, 2.2) dd (7.7, 2.2) d (8.8) dd (12.1, 6.6) m m dd (15.4, 6.6)

3.09, 2.24, 4.18, 2.11, 1.61, 1.42, 2.39, 2.14,

dd (18.7, 7.7) d (18.7) d (7.7) m dd (13.2,6.6) t (13.2) m m

3.49,

s

d (5.5) s dq (14.3, 7.7) dq (14.3, 8.8) m m

3.03, 2.28, 4.30, 1.86, 1.81, 1.61, 1.71, 5.22,

1.22,

m

5.42,

dt (15.4, 7.7)

m t (6.6) dq (14.3, dq (14.3, t (7.7) dq (14.3, dq (14.3, t (7.7) m t (7.7)

1.97, 0.93, 1.79, 1.67, 0.82, 1.57, 1.47, 0.85, 2.10, 0.92,

dq (7.7, 7.7) t (7.7) m dd (14.3, 7.7) t (7.7) m dq (14.3, 6.6) t (6.6) m d (6.6)

m m m t (6.6) m m t (6.6) m dq (14.3, 7.7) t (6.6) d (6.6)

s dd (14.3, 5.5) dd (14.3, 7.7) m m m m

1.22, 0.86, 1.94, 1.87, 0.75, 1.79, 1.69, 0.91, 1.22, 0.82,

0.98, 1.32, 1.24, 0.83, 1.75, 1.65, 0.83, 1.78, 1.46, 0.88, 0.88,

5.93, 2.48, 2.31, 1.68, 1.24, 1.08, 1.24, 1.24, 0.85, 2.27,

m t (6.6) q (7.7)

1.04, 0.83,

t (7.7) d (6.6)

(J in Hz)

7.7) 7.7) 7.7) 7.7)

(J in Hz)

Spectra were recorded at 600 MHz for 1H NMR using residual solvent signals as the internal standard. bδH 5.59 (s, 3-OH), 5.18 (s, 6-OH). COOH: δH 12.34 (br s).

a c

601

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Table 2. 13C NMR Data for Compounds 1−5a 1

a

2

3

4

5

pos.

δC

mult

δC

mult

δC

mult

δC

mult

δC

mult

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

172.2, 121.1, 159.2, 88.1, 64.5, 65.0, 42.0, 29.1, 36.9, 28.6, 22.4, 14.0, 26.6, 7.1, 21.0, 9.3, 19.6,

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

172.2, 121.1, 159.3, 88.0, 64.1, 65.3, 38.6, 34.7, 32.4, 28.2, 22.5, 14.0, 26.7, 7.1, 21.3, 9.4, 25.1, 10.3,

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

175.2, 39.0, 72.5, 93.8, 57.2, 79.9, 44.8, 47.7, 132.2, 131.6, 25.0, 13.9, 30.3, 7.5, 35.8, 8.2, 41.3, 20.4,

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

175.2, 37.8, 73.6, 94.0, 56.6, 81.3, 46.5, 32.6, 42.4, 34.5, 20.9, 15.3, 30.4, 7.5, 34.2, 9.3, 14.6,

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

171.0, 31.9, 141.5, 123.0, 107.6, 152.8, 35.7, 32.0, 34.9, 28.6, 22.4, 14.0, 17.4, 14.8, 19.5,

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

Spectra were recorded at 150 MHz for 13C NMR using residual solvent signals as the internal standard.

calculations (Supporting Information Figure SC1),20 the spatial distances between H-2 and H-3 and H3-16 were potentially close enough to observe NOE correlations. Conversely, in the stable conformers for the 4S/5S/6R diastereomer, H-2 and H-3 were spatially distant from H3-16, and no NOE correlations would be expected. The observed NOE correlations between both H-2 and H-3 and H3-16 in the NOESY spectrum were consistent with the molecular model studies of the former isomer. Accordingly, the absolute configuration for 1 is proposed as 4S/5R/6S. The configuration of C-8 was not determined. The molecular formula of plakortoxide B (2) was determined as C18H30O3 from the HRESIMS data. The similar IR, UV, and 1 H and 13C NMR spectra revealed that 2 is an analogue of 1. A careful comparison of the NMR data for compounds 1 and 2

Figure 1. Selected COSY and HMBC correlations for compounds 1− 5.

Figure 2. Key NOESY correlations for compounds 1−4. 602

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and 0.92 (3H, d, J = 6.6 Hz, H3-18) observed in the 1H NMR spectrum indicated that 3 was possibly an analogue of simplextones A and B.12 After careful comparison and examination of NMR data for 3 with simplextone A, the noticeable difference was found to be that two methylenes in the alkyl chain of simplextone A were replaced by a double bond [δH 5.22 (1H, dd, J = 15.4, 6.6 Hz, H-9); 5.42 (1H, dt, J = 15.4, 7.7 Hz, H-10)] at C-9 in 3, which was confirmed by COSY correlations of H-8/H-9/H-10/H2-11/H3-12 and HMBC correlations of H-9 and H-10 with C-8 [δC 47.7 (CH)] and H3-12 with C-10 (Figure 1). The geometry of the double bond was assigned as E owing to the large coupling constant value (JH‑9/H‑10 = 15.4 Hz). The NOESY correlations between H-3 (δH 4.30) and Ha-13 (δH 1.79) and H3-14 (δH 0.82) suggested that these protons were on the same side of the γ-lactone ring. Also, the NOE correlations between H-5 (δH 1.86) and H-8 (δH 1.71), H2-15 (δH 1.47, 1.57), and H3-18 (δH 0.92) (Figure 2) suggested their cis relationship on the cyclopentane ring. Furthermore, the NOE correlations observed between H-5 and H-3 and H3-14 and between H2-13 and H3-18 indicated the same relative configuration between the conjoined bicyclic systems as for simplextones A and B.12 The absolute configuration of 3 was determined by comparing the observed ECD spectrum with those predicted using the TD-DFT theory method at the RB3LYP/DGDZVP level (Figure 4 and Figures SC3 and SC4). The measured ECD spectrum with a strong positive CE at 191 (Δε 12.55) nm and two weak CEs at 195 nm (Δε 0.94) and 200 nm (Δε −1.27), respectively, for 3 was in near agreement with that of the calculation for the 3S/4S/5S/6R/8R/17R configuration, in contrast to that of its enantiomer 3R/4R/5R/ 6S/8S/17S (Figure 4a and Figures SC3 and SC4). Thus, the absolute configuration of 3 was established as 3S, 4S, 5S, 6R, 8R, 17R in association with biogenetic considerations and the NOESY relationships. Simplextone D (4) has the molecular formula C17H30O4 based on the HRESIMS data. In the 1D NMR spectra of 4, three characteristic triplet methyl proton signals [δH 0.83 (t, J = 6.6 Hz, H3-14); 0.88 (t, J = 6.6 Hz, H3-16); 0.83 (t, J = 6.6 Hz, H3-12)] and one doublet methyl proton signal [δH 0.88 (d, J = 6.6 Hz, H3-17)], together with the carbon signals from C-1 to C-9 and from C-13 to C-16 (Table 2), indicated its structural similarity to 3. Analysis of its 2D NMR spectra (COSY, HMQC, and HMBC) showed that the main differences in 4 occurred at the C-8 and C-9 positions. A doublet methyl group

Figure 3. Experimental CD spectrum of plakortoxide A (1) overlaid with calculated spectra for the candidate stereostructures.

(Tables 1 and 2) suggested that they had the same basic skeleton including the 4-ethyl butenolide and ethyl epoxide moieties, but differed in the alkyl chain, in which the doublet methyl group in 1 was replaced by an ethyl group [δH 1.22 (2H, m, H2-17), 0.82 (3H, t, J = 7.7 Hz, H3-18); δC 25.1 (CH2, C17), 10.3 (CH3, C-18)] in 2, consistent with the molecular formula difference between 1 and 2. The ethyl group at C-8 was confirmed by the HMBC correlations between H3-18 and both C-8 and C-17 (Figure 2). The crucial NOE correlations between H-5 and H2-7 and between both H-2 and H-3 and H316, as well as the CD spectrum with a strong negative Cotton effect at 207 nm (Figure 2 and Supporting Information Figure S55), of 2 suggested that the absolute configuration of 2 was the same as that of 1. The molecular formula of simplextone C (3) was assigned as C18H30O4 with four degrees of unsaturation on the basis of HRESIMS data. Its IR spectrum revealed the presence of hydroxy (3391 cm−1), γ-lactone carbonyl (1746 cm−1), and double-bond (1697 cm−1) groups. The 13C NMR and DEPT data (Table 2) displayed 18 carbons, including four methyl, five methylene, six methine, and three quaternary carbons. One carbonyl [δC 175.2 (C, C-1)] and two olefinic carbons [δC 132.2 (CH, C-9), 131.6 (CH, C-10)] could be easily recognized, and thus two ring systems were necessary to fulfill the degrees of unsaturation for 3. In addition, the characteristic methyl proton signals at δH 0.82 (3H, t, J = 7.7 Hz, H3-14), 0.85 (3H, t, J = 6.6 Hz, H3-16), 0.93 (3H, t, J = 7.7 Hz, H3-12),

Figure 4. Experimental CD spectra of simplextone C (3) (a) and simplextone D (4) (b) overlaid with calculated spectra for the candidate stereostructures. 603

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HeLa (cervical carcinoma), A-549 (lung carcinoma), and BEL7402 (hepatic carcinoma), using the MTT method22 with adriamycin as the positive control (Table 3). Compounds 8, 10,

at C-8 and the alkyl chain C-10/C-11/C-12 attached to C-9 differed from 3. This conclusion was supported by the HMBC correlations from H3-17 to C-7 [δC 46.5 (CH2)], C-8 [δC 32.6 (CH)], and C-9 [δC 42.4 (CH)] and the HMBC correlation from H-9 [δH 2.14 (1H, m)] to C-10 [δC 34.5 (CH2)]. The NOE correlations between H-5 and H-3, H3-14, H-8, H2-15, H2-10, and H2-11 and between H-9 and H3-17 and the experimental and calculated ECD spectra for compound 4 (Figure 4b and Figures SC5 and SC6) supported the same relative and absolute configuration as for 3 apart from 8S in 4, which is due to a change in substituent priority at C-8. Plakorsin D (5) has the molecular formula C15H24O3, deduced from the negative ion HRESIMS spectrum, indicating four degrees of unsaturation. The 1H NMR spectrum of 5 exhibited characteristic methyl signals for Plakortis polyketides at δH 0.85 (3H, t, J = 6.6 Hz, H3-12), 1.04 (3H, t, J = 7.7 Hz, H3-14), and 0.83 (3H, d, J = 6.6 Hz, H3-15), and its 13C NMR and DEPT spectra (Table 2) revealed a total of 15 carbon signals, including three methyl, six methylene, two methine, and four quaternary carbons. The carbonyl group [δC 171.0 (C, C1)] and two double bonds [δC 107.6 (CH, C-5), 123.0 (C, C4), 141.5 (C, C-3), 152.8 (C, C-6)] consumed three degrees of unsaturation, allowing us to assign 5 as a monocyclic structure. Furthermore, the COSY correlations between H3-12 and H2-11 and between H2-9 and H-8 with further coupling to H3-15 and H2-7, as well as HMBC correlations from H2-9 to C-10 and C11 (Figure 1), established a 2-methylhexyl moiety. The presence of one isolated ethyl group was evident from the COSY correlations between H3-14 and H2-13, and the downfield broad singlet proton signal at δH 12.34 observed in its 1H NMR spectrum indicated the presence of a carboxylic acid. A furan ring was suggested by the polarized nature of the double bonds, along with the need to incorporate an extra oxygen and a ring into the structure. In the HMBC spectrum of 5, the correlations from H2-2 [δH 3.49 (s)] to C-1, C-3, and C4 and from H3-14 to C-4 defined acetic acid and ethyl groups at C-3 and C-4, respectively, and the correlations from H-7 [δH 2.48 (dd)] to C-5 and C-6 supported the isoheptyl group attachment at C-6. Thus the structure of 5 was designated as plakorsin D. The 1H and 13C NMR spectroscopic data of 7 were identical to those of woodylide B.13 However, the absolute configuration of woodylide B was not determined. Thus, using the modified Mosher’s method,21 compound 7 was reacted with the (R)(−)- and (S)-(+)-MTPA chlorides to give the (S)-MTPA ester (7a) and the (R)-MTPA ester (7b), respectively. A consistent distribution of positive and negative ΔδH values around C-3 allowed the assignment of the S-configuration for C-3 (Figure 5). The cytotoxicities of compounds 1−11 were evaluated against four human tumor cell lines, K562 (erythroleukemia),

Table 3. Cytotoxicities against HeLa, K562, and A-549 Cell Lines for Compounds 1−11a IC50 (μM) compound 1 2 3 4 5 6 7 8 9 10 11 adriamycinb

HeLa 31.5 >50 29.0 >50 >100 15.5 15.9 4.7 31.2 4.2 2.6 0.6

K562

± 2.9 ± 2.0

± ± ± ± ± ± ±

1.2 1.1 0.5 2.4 0.4 0.3 0.0

>50 >100 19.4 >100 >100 23.8 20.0 2.2 49.2 5.3 0.8 0.3

A-549

± 1.0

± ± ± ± ± ± ±

1.2 1.9 0.2 4.2 0.5 0.1 0.0

>100 >100 40.4 >100 >100 29.3 23.6 >100 >100 >100 >100 0.2

± 3.5

± 2.5 ± 1.2

± 0.0

a

Data represent mean values of three independent experiments and were determined by the MTT method. bPositive control.

and 11 exhibited potent cytotoxic activities against the HeLa and K562 cell lines with IC50 values ranging from 0.78 to 5.32 μM. All of the compounds tested were inactive against the BEL7402 cell line (IC50 value >100 μM). In addition, the inhibitory activities for compounds 1−9 against the receptor tyrosine kinase c-Met,23 an attractive therapeutic target for tumors, were also tested with SU11274 as the positive control. Compound 3 showed significant inhibitory activity against the c-Met kinase target with 57.4% and 34.3% inhibition at concentrations of 10 and 1 μM, respectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Jasco P-1020 digital polarimeter. UV spectra were measured on a Beckman DU640 spectrophotometer. CD spectra were obtained on a Jasco J-810 spectropolarimeter. IR spectra were recorded on a Nicolet NEXUS 470 spectrophotometer using KBr disks. 1D and 2D NMR spectra were recorded on a JEOL JNMECP 600 spectrometer using residual solvent signals (for DMSO-d6: δH 2.50 and δC 39.50 ppm; for CDCl3: δH 7.27 and δC 77.00 ppm) as reference standards. HRESIMS was performed on a Micromass Q-TOF Ultima Global GAA076 LC-MS spectrometer. Semipreparative HPLC utilized an ODS column [YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 1.5 mL/ min]. Silica gel (200−300 mesh, Qingdao, China) was used for column chromatography, and precoated silica gel plates (GF254, Qingdao, China) were used for TLC. Animal Material. The sponge Plakortis simplex (Schulze, 1880), with a feather of thinly encrusting, smooth, black-brownish crust with regularly distributed tiny holes on the surface, was collected from the coral-reef regions near YongXing Island (16°50′ N, 112°20′ E) in the South China Sea, in June 2009, at a depth of about 20 m, and frozen immediately after collection until it was examined. The sponge was identified by one of the authors (N.J.d.V.). A voucher specimen (No. XS2009-08) is deposited in the School of Medicine and Pharmacy, Ocean University of China, People’s Republic of China. Extraction and Isolation. The frozen specimen (6.9 kg, wet weight) was minced and extracted with MeOH three times (each time, 3 days) at room temperature (rt). The combined solutions were concentrated in vacuo, and the concentrated extract was subsequently desalted by redissolving with MeOH to yield a residue (330.0 g). This residue was chromatographed on a silica gel column eluting with an acetone−petroleum ether solvent gradient system (from 0:10 to 10:0,

Figure 5. ΔδH values (ΔδH = δS − δR) obtained for (S)- and (R)MTPA esters of compound 7. 604

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dd, J = 16.5, 10.45 Hz, Ha-2), 2.58 (1H, dd, J = 16.5, 3.3 Hz, Hb-2), 2.02 (1H, m, Ha-13), 1.95 (1H, m, Hb-13); ESIMS m/z 567.2 [M + Na]+. Cytotoxicity Assay. The cytotoxicities of compounds 1−11 against K562, HeLa, A-549, and BEL-7402 human tumor cells were determined by the MTT method.21 Cells were plated in 96-well plates for 24 h before treatment and continuously exposed to different concentrations of compounds for 72 h. Adriamycin was used as a positive control. Results are expressed as the concentration that inhibits 50% control growth after the incubation period (IC50). Experiments were repeated three times and carried out in triplicate. ELISA Kinase Assay. The tyrosine kinase activities were evaluated by an enzyme-linked immunosorbent assay (ELISA) method.22 In general, the ATP solution (50 μL, 10 μM) diluted in kinase reaction buffer (50 mM HEPES, pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was added to each well in 96-well plates. Various concentrations of tested compounds diluted in DMSO (10 μL, 1%, v/v) were then added to each reaction well, with DMSO (1%, v/v) as the negative control and SU11274 (1 μM) as the positive control. The kinase reaction was initiated by the addition of the buffered tyrosine kinase solution (40 μL) (Cell Signaling Technology). After incubation for 60 min at 37 °C, the plate was incubated with anti-phosphotyrosine (PY99) antibody and horseradish peroxidaseconjugated goat anti-mouse IgG for 30 min at 37 °C. Finally, a solution (100 μL) containing H2O2 (0.03%) and o-phenylenediamine (2 mg/mL) in citrate buffer (0.1 mM) was added, and samples were again incubated at rt until color emerged. The reaction was terminated by the addition of an H2SO4 solution (50 μL, 2 M), and the plate was read using a multiwell spectrophotometer (VERSAmax, Molecular Devices) at 490 nm. The inhibition rate (%) was calculated using the following equation:

v/v) to afford eight fractions (Fr.1−Fr.8). Fr.2 (20.2 g) was subjected to a Sephadex LH-20 column eluting with petroleum ether−CHCl3− MeOH (5:5:1, v/v), to give two subfractions, Fr.2-1 and Fr.2-2. Fr.2-2 (10.2 g) was further separated on an ODS reversed-phase silica column followed by stepwise gradient elution with MeOH−H2O (1:4, 2:3, 3:2, 4:1, and 10:0, v/v), and the portion (2.1 g) that eluted with MeOH−H2O (3:2, v/v) was again chromatographed on a silica column eluting with petroleum ether−acetone (35:1, v/v) and finally purified by semipreparative HPLC (ODS; 5 μm, 250 × 10 mm; MeOH−H2O, 80:20, v/v; 2 mL/min) to afford compounds 5 (12.0 mg), 10 (3.0 mg), and 11 (20.0 mg). Also, Fr.3 (21.2 g) and Fr.4 (18.2 g) were separated into two subfractions, Fr.3-1 and Fr.3-2 as well as Fr.4-1 and Fr.4-2, by Sephadex LH-20 column chromatography eluting with petroleum ether−CHCl3−MeOH (5:5:1, v/v). The subfraction Fr.3-2 (10.8 g) was chromatographed on ODS and silica gel columns and by semipreparative HPLC to give compounds 6 (68.0 mg) and 7 (5.5 mg), while compounds 1 (32.0 mg), 2 (5.5 mg), 3 (11.0 mg), 4 (10.0 mg), 8 (10.0 mg), and 9 (12.0 mg) were prepared from Fr.4-2 (9.8 g) using a similar protocol to that for Fr.2-2. Plakortoxide A (1): colorless oil; [α]25D −4.0 (c 0.714, MeOH); UV (MeOH) (log ε) λmax 207 (3.95); CD (c 0.0018 M, MeOH) λmax (Δε) 207.5 (−25.72) nm; IR (KBr) νmax 3420, 2966, 1759, 1696, 1470, 1401, 1401, 1030, 926, 820, 750, 645 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; ESIMS m/z 303.2 [M + Na]+; HRESIMS m/z 303.1933 [M + Na]+ (calcd for C17H28O3Na, 303.1931). Plakortoxide B (2): colorless oil; [α]25D −4.6 (c 0.357, MeOH); UV (MeOH) (log ε) λmax 207 (3.95); CD (c 0.0017 M, MeOH) λmax (Δε) 207.5 (−13.07) nm; IR (KBr) νmax 3416, 2959, 1760, 1697, 1467, 1400, 1381, 1217, 1120, 944, 926, 820, 750, 670, 657 cm−1; 1H and 13 C NMR data, see Table 1 and Table 2; ESIMS m/z 317.2 [M + Na]+; HRESIMS m/z 317.2088 [M + Na]+ (calcd for C18H30O3Na, 317.2087). Simplextone C (3): colorless oil; [α]25D −0.7 (c 0.714, MeOH); UV (MeOH) (log ε) λmax 192 (3.95); CD (c 0.0016 M, MeOH) λmax (Δε) 191 (12.55), 195 (0.94), 200 (−1.27) nm; IR (KBr) νmax 3391, 2951, 1746, 1697, 1468, 1266, 1087, 1030, 943, 751, 646 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; ESIMS m/z 333.3 [M + Na]+; HRESIMS m/z 333.2035 [M + Na]+ (calcd for C18H30O4Na, 333.2036). Simplextone D (4): amorphous powder; [α]25D +26.5 (c 0.714, MeOH); UV (MeOH) (log ε) λmax 196 (3.91); CD (c 0.0012 M, MeOH) λmax (Δε) 193.5 (3.5), 216 (0.88), 226 (−0.65) nm; IR (KBr) νmax 3446, 2953, 2831, 1746, 1697, 1468, 1030, 672 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; ESIMS m/z 321.2 [M + Na]+; HRESIMS m/z 321.2028 [M + Na]+ (calcd for C17H30O4Na, 321.2036). Plakorsin D (5): colorless oil; [α]25D −2.6 (c 0.714, MeOH); UV (MeOH) (log ε) λmax 265 (4.11); IR (KBr) νmax 3199, 2573, 1732, 1575, 1458, 1369, 1036,808, 649, 498 cm−1; 1H and 13C NMR data, see Table 1 and Table 2; ESIMS m/z 251.1 [M − H]−; HRESIMS m/ z 251.1649 [M − H]− (calcd for C15H23O3, 251.1642). Preparation of the (S)- and (R)-MTPA Esters (7a and 7b) of Woodylide B (7). To the solution of 7 (2.0 mg) in pyridine (1 mL) were added (R)-(−)-MTPACl (15 μL) and DMAP (4-N,Ndimethylaminopyridine, 50 μg). The mixture was allowed to react for 48 h at rt and then quenched by the addition of H2O (1 mL), followed by extraction with CHCl3. The CHCl3-soluble portion was evaporated under vacuum. The residue was subjected to HPLC (MeOH−H2O, 78:22, v/v) to afford the (S)-MTPA ester (7a) of 7. ESIMS, 1H NMR, and COSY spectra of 7a were measured, and the corresponding δH values were assigned by COSY correlations. (S)MTPA ester (7a): colorless oil; 1H NMR (selected signals, 600 MHz, CDCl3) δH 7.36 (1H, dd, J = 9.90, 3.85 Hz, H-3), 5.28 (1H, s, H-5), 3.60 (3H, s, OMe), 2.92 (1H, dd, J = 15.9, 9.9 Hz, Ha-2), 2.58 (1H, dd, J = 15.9, 3.85 Hz, Hb-2), 2.05 (2H, q, J = 7.7 Hz, H2-13); ESIMS m/z 567.2 [M + Na]+. Compound 7 (2.0 mg) was treated with (S)(+)-MTPACl (15 μL) by the same procedure described above to afford the (R)-MTPA ester (7b) of 7. (R)-MTPA ester (7b): colorless oil; 1H NMR (selected signals, 600 MHz, CDCl3) δ 7.29 (1H, dd, J = 10.45, 3.30 Hz, H-3), 5.26 (1H, s, H-5), 3.67 (3H, s, OMe), 2.94 (1H,

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%inhibition = [1 − (A490 /A490control )] × 100

ASSOCIATED CONTENT

S Supporting Information *

1D/2D NMR and HRESIMS spectra of compounds 1−5 and 7; computational details for 1, 3, and 4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-532-82032323. Fax: +86-532-82033054. E-mail: [email protected], [email protected]. Author Contributions ∥

J. Zhang and X. Tang contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 41076084 and 20975094), Changjiang Scholars and Innovative Research Team in University (PCSIRT, No. IRT0944), and National Innovative Experimental Program (No. 201210423068). Special thanks are given to Prof. M. Y. Geng and her research group (State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, PR China) for the C-met activity tests.

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