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Cite This: J. Org. Chem. 2018, 83, 14175−14180

Ainsliatriolides A and B, Two Guaianolide Trimers from Ainsliaea fragrans and Their Cytotoxic Activities Rui Zhang,†,‡ Chunping Tang,†,§ Hong-Chun Liu,∥ Yongmei Ren,†,‡,⊥ Cheng-hui Xu,∥ Chang-Qiang Ke,†,§ Sheng Yao,†,§ Xun Huang,*,∥ and Yang Ye*,†,‡,§,⊥

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†

State Key Laboratory of Drug Research and Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China § SIMM-CUHK Joint Research Laboratory for Promoting Globalization of Traditional Chinese Medicines, Shanghai 201203, China ∥ Division of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China ⊥ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China S Supporting Information *

ABSTRACT: Ainsliatriolides A (1) and B (2), two guaianolide sesquiterpenoid trimers possessing an unprecedented skeleton, were isolated from Ainsliaea f ragrans. Their structures were elucidated through extensive analysis of spectroscopic data and confirmed by single-crystal X-ray diffraction experiment. Ainsliatriolides A and B are first examples of compound trimerized from guaianolide sesquiterpenoids through two different C−C linkages (type A, 4− 2′/15−14′; type B, 15′-15″). Ainsliatriolide A displayed potent cytotoxicity with an averaged IC50 value of 1.17 μM against four cancer cells.

T

species is distributed in the southern part of China and has long been used for the treatment of phthisis, coughing up blood, diuresis, edema, clearing heat, and removing toxicity.19,20 Some guaianolide sesquiterpenoids have been reported from this plant.17,18 In our efforts to search for cytotoxic sesquiterpenes, a systematic investigation of A. fragrans was carried out, resulting in the isolation of two guaianolide trimers possessing an unprecedented skeleton (Figure 1), along with two structurally related known guaianolide dimers and one monomer. Herein,

he guaianolide sesquiterpenoids are a class of important secondary metabolites of natural products.1−3 Their fascinating structures and diverse biological activities have attracted extensive interest from both natural product and medicinal chemistry chemists.4−14 Cytotoxic guaianolides have shown great potential to treat cancers; for instance, arglabin, isolated from an Artemisia species, has been approved as an antitumor drug in Kazakhstan.4 Thapsigargin, which was obtained from Thapsia garganica and total synthesized by many medicinal chemists, is a promising anticancer compound known for its activity as a potent antagonist for Ca2+-ATPase inhibition even at subnanomolar concentration.5,15 In addition, guaianolide derivatives such as its dimers and trimers also exhibit potent cytotoxic activities. Ainsliadimer A, a guaianolide dimer originally from Ainsliaea macrocephala, was totally synthesized by Lei and found to have strong potential in the development of anticancer and anti-inflammatory therapies through targeting the cysteine 46 of IKK α/β to block NF-κB signaling.7,12 A guaianolide trimer ainsliatrimer A, which was obtained from Ainsliaea f ulvioides, and also total synthesized by Lei, showed potent anticancer activities deduced by the activation of PPARγ.8,10,16 The Compositae family abounds in sesquiterpenes.1−3 Of the family, the genus Ainsliaea is rich in guaianolide sesquiterpenoids.7,8,17,18 Many Ainsliaea species are used as traditional medicines, including Ainsliaea f ragrans Champ. This © 2018 American Chemical Society

Figure 1. Structures of compounds 1 and 2. Received: September 11, 2018 Published: October 22, 2018 14175

DOI: 10.1021/acs.joc.8b02346 J. Org. Chem. 2018, 83, 14175−14180

Note

The Journal of Organic Chemistry Table 1. 1H and

13

C NMR Data for Compounds 1 and 2 in CDCl3 1a

no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′

δH (J, Hz) 3.28, m 3.27, m 2.62, m

3.34, 4.12, 3.12, 2.32, 1.48, 2.61, 2.27,

6.15, 5.50, 5.08, 4.71, 2.18, 2.05,

2.66, 3.23, 4.21, 3.00, 2.06, 2.02, 2.05, 2.01,

m t (9.4) m m m m m

d (3.2) d (3.2) br s br s m m

m dd (11.0, 3.7) dd (11.0, 9.8) m m m m m

2b

δc

no.

40.2 44.6

10′ 11′ 12′ 13′

222.2 51.2 49.5 84.2 43.3 31.9 39.6 150.4 139.1 169.5 121.0

14′ 15′ 1″ 2″

114.2

3″ 4″ 5″ 6″ 7″ 8″

26.2

9″

173.4 141.6 207.8 49.2 51.2 82.9 51.5 21.0

10″ 11″ 12″ 13″ 14″ 15″

δH (J, Hz)

6.15, 5.50, 1.84, 1.70, 1.91, 1.84, 3.06, 2.48, 2.46,

d (3.2) d (3.2) m m m m m m m

2.29, 2.51, 3.94, 2.96, 2.27, 1.41, 2.56, 2.18,

m m t (9.1) m m m m m

6.19, 5.50, 4.96, 4.63, 1.76, 1.59,

d (3.2) d (3.2) br s br s m m

δc

no.

68.7 140.1 170.4 118.7

1 2

36.5 25.2 21.0 39.8 44.9

3 4 5 6 7 8 9

218.3 51.6 46.6 89.0 44.1 31.6

14

38.7

15

148.7 139.1 169.9 120.9

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′

113.3 22.5

35.8

10 11 12 13

9′

δH (J, Hz) 3.06, m 3.07, m 2.71, m

3.24, 4.23, 2.89, 2.31, 1.50, 2.66, 2.16,

6.28, 5.59, 5.13, 4.79, 2.08, 1.90,

3.07, 4.48, 2.66, 1.95, 1.91, 1.92, 1.67,

t (9.3) t (9.3) m m m m m

d (3.2) d (3.2) br s br s m m

m t (10.5) m m m m m

δc

no.

40.0 44.0

10′ 11′ 12′ 13′

222.0 52.7 48.2 83.9 44.2 32.0 39.9 149.6 138.5 169.9 122.1

14′ 15′ 1″ 2″

114.9

3″ 4″ 5″ 6″ 7″ 8″

26.1

9″

149.6 134.8 115.4 79.4 56.9 80.3 50.5 21.5

10″ 11″ 12″ 13″ 14″ 15″

δH (J, Hz)

6.10, 5.43, 1.78, 1.56, 2.51, 2.13, 3.40, 2.15, 2.04,

2.37, 4.19, 2.77, 2.18, 1.56, 2.35, 2.27,

6.16, 5.45, 4.93, 4.89, 2.18, 1.99,

d (3.3) d (3.3) td (13.9, 3.7) m m m m m m

m dd (11.3, 8.8) m m m m m

d (3.4) d (3.4) br s br s m m

δc 67.9 139.9 170.7 118.4 37.1 27.8 46.5 45.2 109.4 95.2 54.5 79.4 43.7 27.6 27.1 146.1 139.1 169.5 120.5 116.3 22.7

36.9

a

Data were recorded at 500 MHz (1H) and 125 MHz (13C). bData were recorded at 600 MHz (1H) and 125 MHz (13C).

we report the isolation and structural elucidation of these two new compounds and their cytotoxic activity. Compound 1 was obtained as a colorless crystal in methanol. The HRESIMS of 1 recording a quasi-molecular ion at m/z 749.3339 [M + H]+ (cal. 749.3326) suggested a molecular formular of C45H48O10 with 22 degrees of unsaturation. The IR spectrum revealed the presence of hydroxyl (3467 cm−1), γ-lactone (1767 cm−1), and double bonds (1643 cm−1). The 13C and DEPT spectra (Table 1) of 1 displayed 45 carbon resonances including 17 methylenes, 13 methines (three oxygenated at δC 89.0, 84.2, 82.9), and 15 quaternary carbons (three ketonic carbonyls at δC 222.2, 218.3 and 207.8; three ester carbonyls at δC 170.4, 169.9 and 169.5). The 1H NMR spectrum of 1 showed three sets of oxygenated methines protons at δH 4.21 (dd, J = 11.0, 9.8 Hz), δH 4.12 (t, J = 9.4 Hz), δH 3.94 (t, J = 9.1 Hz). All of these were indicative of the existence of three sesquiterpenoid moieties (Figure.2). Starting from the proton resonance at δH 4.12 (t, J = 9.4 Hz, H-6), the HMBC correlations of H-6/C-4; H2-9/C-1; H2-13/ C-7, C-11, and C-12; H2-14/C-1, C-9, and C-10; H-1/C-3; H2/C-3 and C-4; H-5/C-10 and C-15, along with the successive 1 H−1H COSY correlations of H-1−H-5−H-6−H-7−H2-8− H2-9 established the structure of unit A (Figure 2). Similarly, the HMBC and 1H−1H COSY correlations also constructed units B and C (Figure 2). The connectivity of units A and B

Figure 2. Key NMR correlations for 1.

was inferred from the 1H−1H COSY of H2-15 with H2-14′. The HMBC correlations of H-5/C-2′, H2-15/C-10′ and H214′/C-4 further confirmed the linkage of 4−2′/15−14′ (type A). Likewise, the connectivity between unit B and unit C was also deduced by the 1H−1H COSY and HMBC correlations. The 1H−1H COSY correlations of H2-15′−H2−15″ revealed a linkage of C-15′−C-15″ (type B). HMBC correlations from H4″ to C-15′ and from H2-15″ to C-4′ further supported such a connection. Thus, the planar structure of 1 was established (Figure 2). 14176

DOI: 10.1021/acs.joc.8b02346 J. Org. Chem. 2018, 83, 14175−14180

Note

The Journal of Organic Chemistry The relative configuration of 1 was determined by the ROESY experiment (Figure S3). The cross-peaks of H-1/H-7, H-5/H-7, H-6/H2-15 in unit A, the correlations of H-5′/H-7′, H-4′/H-6′ in unit B, and the cross-peaks of H-1″/H-7″, H-5″/ H-7″, H-4″/H-6″ in unit C supported the relative configuration of compound 1 as shown in Figure S3. The absolute configuration of C-10′, however, could not be designated from the ROESY correlations. The full structure including the absolute configuration of 1 was finally determined from the Xray single-crystal diffraction experiment [Cu Kα, at 173 K; Flack parameter: 0.12 (4); CCDC 1844828] (Figure.3).

Figure 4. Perspective ORTEP drawing for 2 (displacement ellipsoids are drawn at the 30% probability level).

then two molecules of dehydrozaluzanin C (5a) led to the formations of compounds 3 and 4 via a regular and a heteroDiels−Alder cycloaddition, respectively. The biomimetic total synthesis of 3 and 4 via regular or hetero-Diels−Alder cycloaddition have already been reported.11,13 The remaining α,β-unsaturated carbonyl group of compound 3 enabled a reaction with the cycloaddition and produced an intermediate 1a. Similarly, 4 could also react with one more molecule of 5a through a regular Diels−Alder cycloaddition between the terminal double bond of 4 and the α,β-unsaturated carbonyl group of 5a. Then, in the existence of Lewis acids, 1a lost a molecule of H2O and formed a carbenium ion (1b), which produced 1c by further losing a proton. A reduction of 1c eventually generated ainsliatriolide A. A pathway from 1a to ainsliatriolide B began with the oxidization of 1a to form 2a. Before proton transfer, ketalation took place between the ketol at C-3′ and the hydroxyl at C-4″ first, affording a hemiketal in 2c, and then the ketol at C-3″ and the hydroxyl at C-3′, which was formed in 2c. Ainsliatriolide B was thus produced. The introduction of α-methylene-γ lactone moieties into the guaianolide sesquiterpenoids was believed to be the cause of their cytotoxic activies.23,24 Compounds 1−5 were evaluated for their cytotoxic effects on A-549, HT-29, BEL-7402, and HL-60 cancer cell lines using CCK-8 assay with doxorubicin (DOX) as a positive control (Table 2). The results showed that all compounds showed different degrees of inhibitory activities against the tested cell lines with IC50 values ranging from 0.8 to 16.5 μM. Compared with the monomer 5, the trimeric and dimeric compounds 1−4 exhibited stronger activities on these four cancer cell lines, especially ainsliatriolide A (1). Ainsliatriolide A showed comparative activities with the positive control against HT-29 and BEL-7402 cell lines. In this study, ainsliatriolides A and B are guaianolide trimers characterized with two different linkages (type A, 4−2′/15− 14′; type B, 15′−15″), which were presumably generated by both regular and hetero-Diels−Alder cycloadditions. Given the fact that Diels−Alder cycloaddition reactions catalyzed by Diels−Alderases have widely been realized,25−27 we assume that ainsliatriolides A and B might also be constructed with the help of Diels−Alderases. Only three guaianolide trimers were reported from natural resources previously.8,28 On the other hand, due to the complex structures, the NMR data of trimers

Figure 3. Perspective ORTEP drawing for 1 (displacement ellipsoids are drawn at the 30% probability level).

Compound 2 was achieved as a colorless crystal in acetone. Its molecular formula of C45H48O12 was determined by HRESIMS (m/z 779.3072 [M-H]−, calcd for C45H47O12, 779.3073), responding to 22 degrees of unsaturation. The IR spectrum revealed the presence of hydroxyl (3462 cm−1), γlactone (1767 cm−1), and double bonds (1641 cm−1). The 1D and 2D NMR data of 2 (Table 1) showed high similarities to those of compound 1, suggesting that they could possess the same structural nucleus. The main differences between compounds 1 and 2 were observed for chemical shifts of C3′ (δC 207.8 vs 115.4), C-4′ (δC 49.2 vs 79.4), C-3″ (δC 218.3 vs 109.4), and C-4″ (δC 51.6 vs 95.2), suggesting that two ketonic carbonyls at C-3′ and C-3″ in compound 1 might be replaced by hemiketal or ketal in 2, and at the same time, the oxygenated quaternary carbons in 2 took the place of two methines at C-4′ and C-4″ in compound 1. Actually, it was a big challenge using 1D and 2D NMR to determine both the planar and absolute structure of 2 due to the existence of four oxygenated quaternary carbons at C-3′, C-4′, C-3″, and C-4″. Fortunately, the suitable crystals of 2 were obtained, and the whole structure of 2 was verified by the X-ray single-crystal diffraction experiment [Cu Kα, at 205 K; Flack parameter: 0.17 (7); CCDC 1844853] (Figure 4). Along with these two guaianolide trimers, two structurally related dimers, gochnatiolide B13 (3) and compound 4, which was first reported as a biomimetic synthesis derivative,11 and one monomer zaluzanin C21 (5) were isolated and identified from A. f ragrans. We proposed a possible biosynthetic pathways starting from zaluzanin C (5) for ainsliatriolides A and B given the cooccurrence of compounds 3−5 (Figure 5). The zaluzanin C (5) was first oxidized to form dehydrozaluzanin C22 (5a), and 14177

DOI: 10.1021/acs.joc.8b02346 J. Org. Chem. 2018, 83, 14175−14180

Note

The Journal of Organic Chemistry

Figure 5. Proposed biogenetic pathways for ainsliatriolides A (1) and B (2). detector using a YMC HPLC column (S-5 μm, 12 nm, 250 × 10 mml). HRESIMS spectra of ainsliatriolide A (1) were recorded on a Waters Synapt G2-Si Q-Tof mass detector, and HRESIMS spectra of ainsliatriolide B (2) was recorded on an Agilent G6520 Q-Tof mass detector. 1D and 2D NMR spectra were recorded using a Bruker AVANCE III 500 or 600 MHz instrument. Chemical shifts were reported in ppm (δ) with coupling constants (J) in hertz. D-101 macroporous resin (Shandong Lu Kang Chemical Industrials, Qingdao, Shandong, China), MCI gel CHP20P (75−150 μm, Mitsubishi Chemical Industries, Japan), Econosep C18 60A (50 μm, DIKMA, China), silica gel (100−200 and 300−400 mesh, Qingdao Haiyang Chemical Co., Ltd., China), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography (CC). TLC was carried out on precoated silica gel 60 F254 aluminum sheets (Merck, Germany), and the TLC spots were viewed at 254 nm and visualized by 5% sulfuric acid in alcohol containing 10 mg/mL of vanillin. Plant Material. The whole plants of A. f ragrans were bought from BoZhou City, Anhui Province, China, in November 2016 and identified by Prof. Min-Jian Qin from the China Pharmaceutical University. A voucher specimen (no. 20161121) was deposited at the Herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. The air-dried whole plants of A. f ragrans (50 kg) were powdered and extracted with 95% EtOH (40 L × 3) at room temperature (96 h each). The combined percolates were concentrated under reduced pressure to give a crude residue (2.3 kg). The residue was suspended in water and then partitioned successively with petroleum ether (PE) and CHCl3, affording a PE (680 g), a CHCl3 (440 g), and a water fraction (1.18 kg), respectively. The CHCl3 extract was chromatographed on a D-101 macroporous resin column eluted with EtOH in water in a stepwise manner (20%, 60%, 80%, 95%) to afford three fractions (A−C). Fraction A was applied to an MCI gel column eluted with EtOH in water (50, 55, 60, 65, 70, 75, 80, 95%), yielding six subfractions (A1−A6) according to TLC analysis. A1 was subjected to CC over Sephadex LH-20 (eluted with CHCl3/MeOH = 1:1) to give three subfractions A1A−A1C. Subfraction A1A was applied to CC over Sephadex LH-20 (eluted with MeOH) to give three subfractions A1A1−A1A3. Subfraction A1A1 was purified by preparative HPLC (MeCN/H2O, 30−50%, 0− 120 min, 25.0 mL/min), yielding ainsliatriolides B (2) (43.3 mg, yield 0.00009%). Subfraction A2 was subjected to CC over Sephadex LH20 (eluted with CHCl3/MeOH = 1:1) to afford three subfractions A2A−A2C. A2A was applied to CC over silica gel, eluting with

Table 2. Cytotoxic Effects of Compounds 1−5 on A-549, HT-29, BEL-7402, and HL-60 Cancer Cell Linesa IC50 (μM) compd 1 2 3 4 5 DOX

A549 1.44 10.64 6.40 5.89 12.20 0.11

± ± ± ± ± ±

0.06 0.55 0.56 0.38 0.62 0.04

HT-29 1.24 5.06 2.64 2.94 13.00 0.82

± ± ± ± ± ±

0.13 0.15 0.54 0.13 3.39 0.07

BEL-7402 1.18 5.09 2.65 2.13 9.51 0.54

± ± ± ± ± ±

0.06 0.27 0.40 0.07 1.88 0.06

HL-60 0.80 3.75 2.81 1.76 16.49 0.07

± ± ± ± ± ±

0.10 0.76 0.31 0.12 5.19 0.01

a

The IC50 values against cancer cells were determined by CCK8 assay after incubation with different concentrations of the indicated compounds for 72 h. Values are shown as mean values ± sd from three independent experiments.

were highly overlapped and hard to assign. It is the first time that we have obtained suitable single crystals and confirmed unambiguously their absolute configuration through the singlecrystal X-ray diffraction experiment. Our findings provided not only naturally occurring novel skeletons with cytotoxic activity but also imply the possibility of finding more new linkages generated between guaianolide monomers and further polymerization due to the reactive functional groups existing in the molecules.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a Rudolph Research Analytical Autopol VI 90079 polarimeter (Hackettstown, NJ). UV spectra were measured on a Shimadzu UV-2550 UV−vis spectrophotometer. Crystal data were collected on a Bruker APEX-II CCD diffractometer. Melting points were detected on a SGW X-4 melting point apparatus. IR spectra were recorded with a Thermo Nicolet FTIR IS5 spectrometer. Analytical HPLC and ESIMS spectra were performed on a Waters 2695 instrument with a 2998 PAD coupled with a Waters Acquity ELSD and a Waters 3100 SQDMS detector. Preparative HPLC was performed on a Varian PrepStar instrument with an Alltech 3300 ELSD detector (Columbia, MD, USA) using a Waters Sunfire RP C18 column (5 μm, 30 × 150 mm). Semi-preparative HPLC was performed on a Waters 2690 instrument with a Waters 996 UV 14178

DOI: 10.1021/acs.joc.8b02346 J. Org. Chem. 2018, 83, 14175−14180

Note

The Journal of Organic Chemistry CH2Cl2/acetone (50:1, 30:1, 20:1, 10:1, 6:1, 4:1, 1:1, 1;2, 1:4) to give eight subfractions A2A1−A2A8. A2A6 was purified with CC over silica gel, eluting with PE/EA (3:1, 2:1, 1:1, 2:3, 1:2, 1:4) to yield five subfractions A2A6A−A2A6F. A2A6D was applied to CC over silica gel, eluting with CH2Cl2/MeOH (100:1, 80:1, 60:1, 40:1, 20:1) to afford four subfractions A2A6D1−A2A6D4. A2A6D4 was finally purified with semipreparative HPLC (MeCN/H2O, 40−65%, 0−60 min, 3.0 mL/min) to yield ainsliatriolide A (1) (6.2 mg, yield 0.00001%). Subfraction A3 was subjected to CC over silica gel, eluting with PE/acetone (15:1, 12:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4) in a stepwise manner to give 10 subfractions A3A−A3J. A3F was applied to CC over silica gel eluting with CH2Cl2/acetone (40:1, 30:1, 20:1, 10:1, 8:1,6:1, 4:1, 2:1, 1;2, 1:4) to obtain nine subfractions A3F1−A3F9. A2F6 was then purified by preparative HPLC (MeCN/ H2O, 20−40%, 0−120 min, 25.0 mL/min) to yield zaluzanin C (5) (400 mg, yield 0.0008%). Fraction B was subjected to CC over Sephadex LH-20 (eluted with CHCl3/MeOH = 1:1) to give four subfractions B1−B4. Subfraction B2 was applied to CC over silica gel, eluting with PE/acetone (25:1, 20:1, 15:1, 12:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4) to afford 14 subfractions B2A−B2N. B2L was subjected to CC over silica gel, eluting with CH2Cl2/acetone (30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 2:1, 1;2, 1:4) to give eight subfractions B2L1−B2L8. B2L3 was applied to a C18 column eluting with MeOH/H2O (50%, 55%, 60%, 65%, 70%, 80%, 90%) to yield eight subfractions B2L3A−B2L3H. B2L3C was then purified by preparative HPLC (MeCN/H2O, 25−45%, 0−120 min, 25.0 mL/min) to afford gochnatiolide B (3) (160 mg, yield 0.0003%). B2N was subjected to a C18 column, eluting with MeOH/H2O (50%, 55%, 60%, 65%, 70%, 80%, 90%) to obtain four subfractions B2N1−B2N4. B2N2 was then applied to preparative HPLC (MeCN/H2O, 30−50%, 0−120 min, 25.0 mL/min) to yield 4 (40 mg, yield 0.00008%). Ainsliatriolide A (1): colorless bulk crystals; mp 226−227 °C; [α]20D −34 (c 0.1, MeOH); HRESIMS m/z 749.3339 [M + H]+ (calcd for C45H49O10 749.3326); UV (MeOH) λmax (log ε) 205 (5.27), 244 (4.56); IR (KBr) νmax 3467, 2928, 2860,1767, 1696, 1643, 1444, 1404, 1260, 1142, 999, 948, 817 cm−1; 1H and 13C NMR data see Table 1. Ainsliatriolide B (2): colorless bulk crystals; mp >280 °C; [α]20D −40 (c 0.1, MeOH); HRESIMS m/z 779.3072 [M − H]− (calcd for C45H47O12 779.3073); UV (MeOH) λmax (log ε) 206 (5.56); IR (KBr) νmax 3462, 2928, 2865,1767, 1696, 1641, 1444, 1403, 1259, 1138, 1000, 940, 815 cm−1; 1H and 13C NMR data, see Table 1. Cytotoxicity Assays. The antiproliferative activities of the compounds were evaluated against four human cancer cell lines, A549 (nonsmall cell lung cancer), HT-29 (colorectal cancer), BEL7402 (hepatocellular carcinoma), and HL-60 (leukemia), by the Cell Counting Kit-8 (CCK-8). Briefly, cells seeded into 96-well plates and grown for 24 h were treated with increasing concentrations of compounds and incubated for a further 72 h. At the end of the exposure time, 10 μL of CCK8 (Dojindo, Kumamoto, Japan) was added to each well, and the plates were kept in the incubator for 4 h and then measured at 450 nm using a multiwell spectrophotometer (SpectraMax, Molecular Devices, U.S.A). The inhibition rate was calculated as (1 − A450 treated/A450 control) × 100. The cytotoxicity of compounds was expressed as IC50, determined by the Logit method. Doxorubicin HCl was used as the positive control (Meilun, Dalian, China).

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ainsliatriolides ainsliatriolides X-ray data for X-ray data for

A (1) and B (2); MS and IR spectra of A (1) and B (2) (PDF) compound 1 (CIF) compound 2 (CIF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yang Ye: 0000-0003-1316-5915 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful for the financial support of the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (2015ZX09103002) and the National Natural Science Foundation of China (81573305, 81673327). Part of the work was supported by the International Partnership Program of Chinese Academy of Sciences (153631KYSB20160004).

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REFERENCES

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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.joc.8b02346. X-ray crystallographic analyses of ainsliatriolides A (1) and B (2); X-ray crystallographic data for ainsliatriolides A (1) and B (2); perspective ORTEP drawing for ainsliatriolides A (1) and B (2); key ROESY correlations for ainsliatriolide A (1); spectroscopic data for 14179

DOI: 10.1021/acs.joc.8b02346 J. Org. Chem. 2018, 83, 14175−14180

Note

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DOI: 10.1021/acs.joc.8b02346 J. Org. Chem. 2018, 83, 14175−14180