Dysideanones A–C, Unusual Sesquiterpene Quinones from the South

Article pubs.acs.org/jnp

Dysideanones A−C, Unusual Sesquiterpene Quinones from the South China Sea Sponge Dysidea avara Wei-Hua Jiao,† Ting-Ting Xu,†,‡ Hao-Bing Yu,§ Guo-Dong Chen,⊥ Xiao-Jun Huang,⊥ Fan Yang,† Yu-Shan Li,‡ Bing-Nan Han,† Xiao-Yan Liu,*,† and Hou-Wen Lin*,†,§ †

Key Laboratory for Marine Drugs, Department of Pharmacy, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, People’s Republic of China ‡ Department of Pharmacognosy, Shenyang Pharmaceutical University, Shenyang, 110016, People’s Republic of China § Laboratory of Marine Drugs, Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, People’s Republic of China ⊥ Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou 510632, People’s Republic of China S Supporting Information *

ABSTRACT: Dysideanones A−C (1−3), three unusual sesquiterpene quinones with unprecedented carbon skeletons, were isolated from the South China Sea sponge Dysidea avara. Their structures including absolute configurations were determined by a combination of spectroscopic analyses and calculated ECD spectra. Within the sesquiterpene quinone structures, dysideanones A (1) and B (2) share an unprecedented 6/6/6/6fused tetracyclic carbon skeleton, while dysideanone C (3) possesses an unusual 6/6/5/6-fused tetracyclic core. Dysideanone B (2) showed cytotoxicity against two human cancer cell lines, HeLa and HepG2, with IC50 values of 7.1 and 9.4 μM, respectively.

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structure elucidation, absolute configuration assignments, and bioactivity of the three novel metabolites.

arine sponges of the genus Dysidea have been reported to be a rich source of secondary metabolites with extensive structural diversity. These metabolites comprise bromophenols,1−3 sesquiterpenoids,4 diterpenoids,5 sterols,6 and polychlorinated compounds such as dysidamides,7 dysideathiazoles,8 and chlorinated diketopiperazines.9 Sesquiterpene quinones and hydroquinones, as one of the most important chemical types of Dysidea sponges, have attracted much interest due to their structural diversity and varied biological activities, including antiHIV,10 antibacterial,11 antifungal,12 antioxidative,13 antitumor,14,15 anti-inflammatory,11,16 and protein tyrosine phosphatase 1B (PTP1B) inhibitory activities.17 Our previous chemical investigation of the sponge Dysidea avara led to the isolation of four novel sesquiterpene quinones, dysidavarones A−D.17 Recently Schmalzbauer et al. synthesized dysidavarone A and found this compound showed potent antibacterial activity against Gram-positive bacteria (including methicillin- and multi-drug-resistant Staphylococcus aureus) with MIC50 values of 0.2−9.9 μg/mL.18 Their interesting findings encouraged us to continue searching for biologically active metabolites with related structures. Further chromatographic purification of the minor sesquiterpene quinone components in the extracts of D. avara resulted in the isolation of dysideanones A−C (1−3). Compounds 1 and 2 share a 6/6/6/6-fused tetracyclic carbon skeleton, while compound 3 possesses an unusual 6/6/5/6-fused tetracyclic core. Herein, we reported the © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Following the isolation and characterization of dysidavarones A−D from D. Avara, the minor sesquiterpene quinone components were further subjected to silica gel, Sephadex LH-20, and reversed-phase HPLC to afford dysideanones A−C (1−3). Dysideanone A (1) was obtained as yellowish powder. Its molecular formula was determined as C21H28O2 by the HRESIMS positive ion peak at m/z 313.2166 [M + H]+. The IR spectrum exhibited absorptions for a hydroxy group at 3384 cm−1 and an α,β-unsaturated carbonyl group at 1668 cm−1.16 Analysis of the 13 C and DEPT135 NMR spectra of 1 indicated the presence of 21 carbons, including one carbonyl carbon (δC 185.5), five Received: November 12, 2013 Published: February 18, 2014 346

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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data of 1−3 in CDCl3a 1 position

δC, type

1α 1β 2α 2β 3α 3β 4 5 6α 6β 7a 7b 8 9 10 11a 11b 12 13 14 15α 15β 16 17 17-OH 18 19 20 21 22 23

39.7, CH

a

24.7, CH2 119.0, CH 142.4, C 37.6, C 36.6, CH2 27.6, CH2 43.6, CH 41.4, C 47.3, CH 18.0, CH3 15.4, CH3 19.6, CH3 16.0, CH3 46.7, CH2

δH (J in Hz)

2 δH (DMSO-d6)

3 δH (J in Hz)

32.9, CH 2.00, m 2.70, m 2.17, m 5.22, br s

1.83, m 2.63, m 2.06, m 5.16, br s

1.34, m 1.74, d (12.7) 1.45, m

1.26, m 1.67, dt (12.5, 2.5) 1.40, m

1.50, m

1.37, m

1.72, d (12.5) 1.60, br s

1.61, d (12.5) 1.55, br s

0.85, s 0.87, d (5.9) 0.73, s 2.49, d (11.7) 2.16, d (11.7)

0.79, s 0.83, d (6.0) 0.66, s 2.38, d (11.5) 2.14, d (11.0)

162.4, C 70.2, C 149.6, CH 128.2, CH 185.5, C 124.1, CH

δC, type

7.01, d (10.1) 6.19, dd (10.1, 1.9)

5.57, s 7.01, d (10.0) 6.05, d (10.5, 2.0)

5.97, d (1.9)

5.90, br s

33.8, CH2 32.9, CH2 158.8, C 39.2, C 37.5, CH2 26.9, CH2 43.6, CH 34.7, C 53.9, CH 103.7, CH2 20.6, CH3 15.7, CH3 14.2, CH3 40.1, CH2

δC, type 21.5, CH2

2.87, m 1.02, m 2.52, m 2.23, br d (14.0) 2.57, m

1.73, m 1.58, m 1.47, m 1.26, m 1.12, d (11.0) 4.57, s 4.55, s 1.11, s 0.93, d (6.5) 0.68, s 2.72, d (19.0) 1.82, dd (19.0, 4.5)

27.6, CH2 121.1, CH 143.4, C 37.5, C 34.4, CH2 28.4, CH2 46.0, C 46.5, C 51.2, CH 18.0, CH3 18.3, CH3 24.0, CH3 21.1, CH3 47.3, CH2

142.9, C 141.7, C

168.3, C 81.1, C

187.7, C 106.9, CH 158.2, C 182.3, C 65.0, CH2 13.9, CH3

146.3, CH 128.6, CH 186.6, C 121.8, CH

5.81, s

δH (J in Hz)

δH (DMSO-d6)

1.50, m 1.61, m 2.02, m 2.10, m 5.25, br s

1.44, m 1.88, m 1.92, m 2.00, m 5.15, br s

1.88, td (14.9, 4.5) 1.57, dd (10, 4.5) 1.61, m 1.96, dd (14.3, 2.0)

1.92, m 1.44, m 1.47, m 1.90, m

2.26, dd (12.2, 1.9) 1.59, br s

2.24, m 1.50, d (1.0)

0.99, s 0.68, s 1.00, s 2.94, dd (18.6, 2.6) 2.23, d (18.6)

0.91, s 0.55, s 0.89, s 2.77, dd (18.0, 2.5) 2.17, d (18.0)

6.95, d (10.0) 6.10, dd (10.0, 1.7)

5.45, s 6.99, d (10.0) 5.96, d (10.0, 1.5)

5.96, br s

5.85, br s

3.98, m 1.48, t (7.0)

Assignments of the 13C and 1H signals were made on the basis of HSQC spectroscopic data.

C6−C7−C8−C13, while the HMBC correlations of H-1/C-5, C-9, and C-10, H3-12/C-5, C-6, and C-10, H-6β/C-8 and C-10, H2-7 and H3-13/C-9, and H3-14/C-8, C-9, and C-10 suggested the linkage of ring B. Thus a bicyclic moiety was revealed with four methyl groups (H3-11, H3-12, H3-13, and H3-14) attached at C-4, C-5, C-8, and C-9, respectively. The COSY correlation of H-18/ H-19 and HMBC correlations of H-18/C-16 and C-20, H-19/ C-17 and C-21, and H-21/C-17 and C-19 indicated the presence of ring D. The HMBC correlations of H2-15/C-8, C-9, C-10, C-16, C-17, and C-21 supported the linkage of C-9 and C-16 via the methylene CH2-15 between rings B and D. Moreover, the HMBC correlations of H2-2 and H-10/C-17 connecting rings A and D via a new carbon−carbon bond C1−C17 indicated the formation of the new six-numbered ring C (Figure 1). Thus, the planar structure of 1 was elucidated as shown in Figure 1. The large coupling constant between H-1 and H-10 (J = 12.5 Hz) and the NOESY correlations of H-1/H3-12 and H3-14 indicated the axial orientations of these protons and methyls and also revealed the trans fusion of rings A/B/C.19 The NOESY correlations from H-8 to H-6α, H-10, and H-15α suggested the four protons were positioned on the same face, while the NOESY correlations of H3-13/H3-14, H-6β/H3-12, and H-15β/H3-13 indicated these methyls and protons were positioned on the other face. The NOESY experiment in DMSO-d6 showed 17-OH had correlations with H-8, H-10, and H-15α, indicative of the α-orientation of the hydroxy group as shown in Figure 2.

quaternary carbons, seven methine carbons, four methylene carbons, and four methyl carbons (Table 1). The 1H NMR spectrum of 1 in CDCl3 indicated the presence of three singlet methyls, one doublet methyl, and four olefinic protons. Additionally, one hydroxy proton was also observed at δH 5.57 (s) in the 1H NMR spectrum recorded in DMSO-d6 (Table 1 and Figure S2). The COSY cross-peaks of H-1/H-10, H-1/H-2α, H-2β/H-3, and H-3/H3-11 delineated the spin system C10−C1−C2−C3− C4−C11 (Figure 1). The HMBC correlations of H3-11/C-3, C-4,

Figure 1. Key COSY and HMBC correlations of 1−3.

and C-5, H3-12/C-4, C-5, and C-10, and H-1 and H-2β/C-10 established ring A. Meanwhile the COSY correlations of H-6α/ H2-7, H2-7/H-8, and H-8/H3-13 indicated the connectivity of 347

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(δC 65.0 and 13.9), as well as the absence of the methyl group CH3-11 in 2 (Figure S15). The extra methylene and exomethylene groups were determined at C-3 and C-11 by the COSY correlation of H2-2/H2-3 and HMBC correlations of H2-11/C-3, C-4, and C-5 as well as H2-3/C-1, C-4, and C-5, respectively (Figure 1). Furthermore, the HMBC correlations of H-10/C-17, H-19/C-17, C-20, and C-21, and H-15α/C-16, C-17, and C-21 indicated that the two carbonyls were located at C-18 and C-21, respectively. Additionally, the HMBC correlation of H2-22/C-20 and NOESY correlation of H-19/ H2-22 placed the O-ethyl group at C-20. The trans fusion of rings A/B/C in 2 was assigned by the coupling constant between H-1 and H-10 (J = 11.0 Hz) and NOESY correlations of H3-12/H3-14 and H-8/H-6α/H-10, and thus its relative configurations from C-1 to C-10 were consistent with those of 1 (Figure 2). The absolute configuration of 2 was determined as 1S,5S,8S,9R,10S by comparison of its experimental and calculated ECD spectra as shown in Figure 4. Dysideanone B (2), however, could possibly

Figure 2. Key NOESY correlations of 1−3.

The absolute configuration of compound 1 was determined by comparison of the observed electronic circular dichroism (ECD) spectrum with time-dependent density functional theory (TD-DFT)-calculated ECD spectra.20−22 The stereostructure of 1 was constructed based on the distance constraints from the key correlations observed in the NOESY spectrum. Using these spectroscopic data, only the stereoisomer as shown and its corresponding mirror image were found to satisfy the observed NOE constraints. Therefore, only two configurations were generated for theoretical calculations to identify the most probable candidate for 1. The optimized conformations were used for the ECD calculations, which were performed with Gaussian 09 (B3P86/6-311++G (2d,p)). The calculated ECD spectra of 1 were compared with the experimental ECD spectrum to determine the most probable configuration. Finally, the Boltzmann-averaged ECD spectrum of 1 was obtained with the aid of Multiwfn 3.2 software (Figure 3). The overall pattern

Figure 4. Comparison of the experimental ECD spectrum with those calculated for the two possible enantiomers (1R,5R,8R,9S,10R and 1S,5S,8S,9R,10S) of 2.

be a byproduct from the extraction process with EtOH as a solvent. The molecular formula of dysideanone C (3) was assigned as C21H28O2, an isomer of 1, by its HRESIMS ion peak at m/z 313.2169 [M + H]+. The 1H and 13C NMR data of 3 were similar to those of 1, except for one methylene (δC 21.5/δH 1.61 and 1.50, CH2-1) and two quaternary carbons at δC 46.0 (C-8) and 81.1 (C-17) (Table 1, Figures S25 and S27). The HMBC correlations of H3-13/C-7, C-8, C-9, and C-17, H2-7/C-17, and H-19 and H-21/C-17 indicated a new carbon−carbon bond between C-8 and C-17 to form the new five-membered ring C between the decalin ring B and ring D (Figure 1). In the NOESY spectrum of 3, correlations of H3-12/H3-14, H-6α/H-10, and H-6β/H3-12 revealed the trans fusion of rings A/B, while the correlations from H3-13 to H3-14 assigned the cis fusion of rings B/C, further supported by the correlations of H3-13/H-15β and H3-14/H-15β. In addition, the NOESY spectrum in DMSO-d6 of 3 showed the correlations from 17-OH to H-10 and H-15α, which established an α-orientation for 17-OH (Figure 2). Comparion of the experimental and calculated ECD spectra of 3 determined its absolute configuration as 5S,8R,9S,10S,17R (Figure 5). There are a series of naturally occurring derivatives of avarone;17,23 however, dysideanones A−C (1−3) are the first examples with unprecedented 6/6/6/6-fused (1 and 2) and 6/6/ 5/6-fused (3) tetracyclic carbon skeletons. Their biosynthetic

Figure 3. Comparison of the experimental ECD spectrum with those calculated for the two possible enantiomers (1R,5R,8R,9S,10R,17R and 1S,5S,8S,9R,10S,17S) of 1.

of the calculated spectrum was in good agreement with the experimental one. Thus, the absolute configuration of 1 was determined as 1S,5S,8S,9R,10S,17S. The molecular formula of dysideanone B (2) was determined as C23H30O3 by its HRESIMS, which corresponded to nine units of unsaturation. The UV absorptions at 230 and 273 nm implied a benzoquinone chromophore. The 1H and 13C NMR spectra of 2 were similar to those of 1, except for the presence of two carbonyl carbons at δC 182.3 and 187.7, an exomethylene carbon at δC 103.7, a methylene carbon at δC 32.9, and an O-ethyl group 348

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volume of CH2Cl2 four times to yield 32 g of a CH2Cl2 solvent extract, which was partitioned between 90% aqueous MeOH and n-hexane to give 12 g of n-hexane fraction and a 20 g aqueous MeOH fraction. Addition of H2O to the aqueous MeOH fraction afforded a 60% aqueous MeOH solution, which was partitioned by CH2Cl2 five times to yield 11 g of a CH2Cl2-soluble fraction. The CH2Cl2-soluble fraction was subjected to a silica gel chromatography column eluted with a gradient of CH2Cl2 and MeOH, yielding three subfractions (D1−D3). Fraction D2 (6.6 g) was passed through an ODS chromatography column eluted with a gradient of aqueous MeOH, size-exclusion chromatography Sephadex LH-20 eluted with CH2Cl2/MeOH (1:1), and then purified by reversed-phase HPLC (YMC-Park Pro C18, 10 × 250 mm, 2 mL/min, 280 nm) with 70% CH3CN, to give dysideanone A (1, 2.6 mg, tR 37.1 min), dysideanone C (3, 2.1 mg, tR 47.8 min), and dysideanone B (2, 5.6 mg, tR 78.2 min). Dysideanone A (1): pale yellow powder; [α]D +2.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.02), 232 (3.78) nm; CD (1.8 × 10−4 M, MeOH), λmax (Δε) 346 (−2.31), 295 (+0.34), 259 (−8.06), 230 (+17.1), and 210 (−12.9) nm; IR (KBr) νmax 3384, 2960, 2926, 2860, 1724, 1668, 1629, 1609, 1461, 1385, 1288, 1019, 811 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 313.2 [M + H]+; HRESIMS m/z 313.2166 [M + H]+ (calcd for C21H29O2, 313.2168). Dysideanone B (2): pale yellow powder; [α]D +82 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (4.01), 230 (3.71), 273 (3.67) nm; CD (2.7 × 10−4 M, MeOH), λmax (Δε), 314 (−0.14), 272 (+7.8), and 210 (−1.19) nm; 1H and 13C NMR data, see Table 1; IR (KBr) νmax 3356, 2925, 2855, 1730, 1650, 1606, 1461, 1383, 1222, 1035, 893 cm−1; ESIMS m/z 355.2 [M + H]+; HRESIMS m/z 355.2271 [M + H]+ (calcd for C23H31O3, 355.2273). Dysideanone C (3): pale yellow powder; [α]D +71 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 203 (4.02), 231 (3.99) nm; CD (2.1 × 10−4 M, MeOH), λmax (Δε) 316 (−0.97), 289 (−4.46), 247 (+34.5), and 215 (−23.4) nm; 1H and 13C NMR data, see Table 1; IR (KBr) νmax 3274, 2959, 2927, 1728, 1669, 1633, 1456, 1386, 1291, 1063, 868 cm−1; ESIMS m/z 313.2 [M + H]+; HRESIMS m/z 313.2169 [M + H]+ (calcd for C21H29O2, 313.2168). Energy Minimization and ECD Calculations. The initial conformations of 1, 2, and 3 were optimized using the MMFF94 method in MarvinSketch 5.8.1 and then the HF/6-31G(d) method in Gaussian09.26 Further optimization at the B3P86/6-31G(d) level led to the final dihedral angles. The optimized conformations were used for the ECD calculations, which were performed with Gaussian 09 (B3P86/ 6-311++G (2d,p)). The solvent effects were taken into account by the conductor-like polarizable calculation model (CPCM, MeOH as the solvent). Cytotoxicity Bioassay. The MTT method was used for in vitro evaluation of the cytotoxic potential of compound 2 against the human cancer cell lines HeLa and HepG2. All the cells were cultured in RPMI1640 or DMEM medium (Hyclone), supplemented with 10% fetal bovine serum (Hyclone) in 5% CO2 at 37 °C. The cytotoxicity assay was performed in 96-well microplates.27 Briefly, adherent cells (100 μL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with an initial density of 1 × 105 cells/mL. Each cancer cell line was exposed to the tested compound at concentrations of 1, 10, 50, and 100 μM in triplicate for 48 h with 5-florouracil as the positive control. The cells in each well were then solubilized with DMSO (100 μL for each well), and the optical density (OD) was recorded at 595 nm. IC50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves.

Figure 5. Comparison of the experimental ECD spectrum with those calculated for the two possible enantiomers (5R,8S,9R,10R,17S and 5S,8R,9S,10S,17R) of 3.

precursor may be avarol (4), which was originally isolated from the title sponge.24 Although dysidavarone A was reported to have potent antimicrobial activity against some Gram-positive bacteria,18 none of the compounds 1−3 showed any antibacterial activity against S. aureus and Bacillus subtilis or antifungal activity against Candida albicans, Rhodothece glutinis, and Aspergillus niger at concentrations as high as 1 mg/mL. Compound 2, however, showed cytotoxic activity with IC50 values of 7.1 and 9.4 μM, respectively, against two human cancer cell lines, HeLa and HepG2, by the MTT method using 5-fluorouracil as a positive control with respective IC50 values of 2.3 and 7.6 μM.25 A scarcity of material hampered the evaluation of the cytotoxicity of dysideanones A (1) and C (3).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation measurements were conducted on an Autopol I polarimeter (No. 30575, Rudolph Research Analytical) with a 10 cm length cell at room temperature. UV and IR (KBr) spectra were recorded on a Hitachi U-3010 spectrophotometer and Jasco FTIR-400 spectrometer, respectively. CD spectra were obtained on a Jasco J-715 spectropolarimeter. 1H, 13C, DEPT135, COSY, HSQC, HMBC, and NOESY NMR spectra were recorded at room temperature on a Bruker Avance DRX600 MHz NMR spectrometer with CDCl3 as the solvent and internal standard. Spectra were referenced to residual solvent signals with resonances at δH/δC 7.26/77.0 for CDCl3 and 2.49/39.5 for DMSO-d6. ESIMS spectra were obtained using a Finnigan MAT 95 spectrometer, and HRESIMS spectra were measured on an Agilent 6210 LC/MSD TOF mass spectrometer. Column chromatography was conducted using precoated silica gel (65 × 250 or 230 × 400 mesh). Analytical thin-layer chromatography (TLC) systems were performed on silica gel 60 F254 plates. Sephadex LH-20 was purchased from Amersham Pharmacia Biotech AB. Purification of the compounds was performed using a Waters Alliance 2695 separation module equipped with a Waters 2998 photodiode array (PDA) detector. All chemicals were of analytical grade; solvents for open column chromatography and MPLC were also analytical grade, whereas solvents for HPLC were chromatographic grade. MPLC and HPLC were performed with columns of 50 and 5 μm ODS, respectively. Animal Material. Samples of Dysidea avara were collected along the coast of Yongxing Island in Xiasha on April 19, 2010. The voucher number for this collection is XD10409, and a voucher sample is maintained at the Laboratory of Marine Drugs, Changzheng Hospital, Second Military Medical University, Shanghai, China. Extraction and Isolation. The animals (2.0 kg, dry weight) were soaked in 95% EtOH repeatedly to give 92.9 g of an EtOH extract. The extract was dissolved in 1 L of H2O and partitioned with the same

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ASSOCIATED CONTENT

S Supporting Information *

Copies of 1D and 2D NMR, HRESIMS, UV, IR, and CD spectra for 1−3. These materials can be accessed free of charge via the Internet at http://pubs.acs.org. 349

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(18) Schmalzbauer, B.; Herrmann, J.; Müller, R.; Menche, D. Org. Lett. 2013, 15, 964−967. (19) Paterson, I.; Dalby, S. M.; Roberts, J. C.; Naylor, G. J.; Guzmán, E. A.; Isbrucker, R.; Pitts, T. P.; Linley, P.; Divlianska, D.; Reed, J. K.; Wright, A. E. Angew. Chem., Int. Ed. 2011, 50, 3219−3223. (20) Berova, N.; Di Bari, L.; Pescitelli, G. Chem. Soc. Rev. 2007, 36, 914−931. (21) Menna, M.; Aiello, A.; D’Aniello, F.; Imperatore, C.; Luciano, P.; Vitalone, R.; Irace, C.; Santamaria, R. Eur. J. Org. Chem. 2013, 3241− 3246. (22) Zou, Y.; Hamann, M. T. Org. Lett. 2013, 15, 1516−1519. (23) Diaz-Marrero, A. R.; Austin, P.; Soest, R. V.; Matainaho, T.; Roskelley, C. D.; Roberge, M.; Andersen, R. J. Org. Lett. 2006, 8, 3749− 3752. (24) Minale, L.; Riccio, R.; Sodano, G. Tetrahedron Lett. 1974, 15, 3401−3404. (25) Carmichael, J.; De Graff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer Res. 1987, 47, 936−942. (26) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.1; Gaussian, Inc.: Wallingford, CT, 2009. (27) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63.

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-21-68383346. Fax: +86-21-58732594. E-mail: [email protected] (X. Y. Liu). *E-mail: [email protected] (H. W. Lin). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Fei Peng at Fujian Institute of Microbiology for his contribution to evaluating the antibacterial and antifungal activities of 1−3. This research was supported by the National Natural Science Fund for Distinguished Young Scholars of China (81225023), the National Natural Science Fund of China (Nos. 41106127, 81072573, 81172978, 81302691, 81373321, and 81202441), Shanghai Subject Chief Scientist (12XD1400200), and National High Technology Research and Development Program of China (863 Projects, Nos. 2011AA09070107 and 2013AA092902). The authors also thank the high-performance computing platform of Jinan University.

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DEDICATION Dedicated to Academician Xin-Sheng Yao, of Shenyang Pharmaceutical University and Jinan University, for his pioneering work on natural products chemistry and traditional Chinese medicines on the occasion of his 80th birthday.

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REFERENCES

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