Structural Investigation and Biological Activity of Sesquiterpene

Mar 1, 2013 - The supplementary crystallographic data for 1, 2, 4, and 5 reported in this paper have been deposited at the Cambridge Crystallographic ...
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Structural Investigation and Biological Activity of Sesquiterpene Lactones from the Traditional Chinese Herb Inula racemosa Yan-Yan Ma, Deng-Gao Zhao, and Kun Gao* State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China S Supporting Information *

ABSTRACT: Five new sesquiterpene lactones, racemosalactones A−E (1−5), along with 19 known sesquiterpene latones (6−24), were isolated from the roots of Inula racemosa. Their structures were elucidated by extensive spectroscopic analysis, and the absolute configuration of 2 was deduced from X-ray diffraction analysis. Compounds 1, 6, 8, 10, 12, 14, and 17 exhibited antiproliferative activities with IC50 values ranging from 0.38 to 4.19 μg/mL against human non-small-cell lung cancer A549, hepatocellular carcinoma HepG2, and human fibrosarcoma HT1080 cells. Compounds 6 and 8 exhibited antiproliferative activities against endothelial cells with IC50 values of 2.4 and 2.5 μg/mL, respectively. Furthermore, compounds 6 and 8 both inhibited endothelial cell tube formation at 1.0 μg/mL. A method for the rapid and straightforward preparative-scale isolation of compound 6 from alantolides is described.

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hytochemical investigations of the Asteraceae (Compositae) family for structurally diverse and biologically significant sesquiterpenoids have been attractive programs of natural products1 and synthesis chemistry.2 The genus Inula comprises about 100 species and is distributed widely in Asia, Europe, and Africa.3 Inula racemosa Hook.f. is an economically and medicinally important perennial herb that has long been used in China, India, and Europe. It is a rich source of sesquiterpene lactones, a widely distributed class of natural products with interesting biological activities. Previous studies have shown that the roots of I. racemosa contain high levels of eudesmane-type sesquiterpene lactones,3,4 in particular alantolactone (6) and isoalantolactone (8).5 Pharmacological studies showed that 6, 8, and their analogues possess a wide spectrum of biological activities, such as antitumor, antifungal, antibacterial, insecticidal, and plant growth regulatory activities.6 As a part of our continuous search for novel sesquiterpene lactones from plants of the Asteraceae family, and in an attempt to learn more about the structure and biological activity relationships among the sesquiterpene lactones of I. racemosa, four eudesmane-type (1−4) and one eremophilane-type (5) sesquiterpene lactone, together with 19 known analogues (6− 24), were isolated from the roots of I. racemosa. Herein, we report the structural elucidation and biological activity of the sesquiterpene lactones from the roots of I. racemosa.

alantolactone (8),4a 11,13-dihydroisoalantolactone (9),7 alloalantolactone (10),4a 3-oxoalloalantolactone (11),6b 5α-epoxyalantolactone (12),4b dihydroepoxyalantolactone (13),4c 4(15)α-epoxyisoalantolactone (14),4b dihydro-4(15)α-epoxyisoalantolactone (15),6b telekin (16),4d isotelekin (17),4a macrophyllilactone E (18),8 3β-hydroxy-11α,13-dihydroalantolactone (19),3 11α-hydroxyeudesm-5-en-8β,12-olide (20),3 11,13-dihydro-2α-hydroxyalantolactone (21),9 11,13-dihydroivalin (22),10 11βH-2α-hydroxyeudesman-4(15)-en-12,8β-olide (23),11 and 11,12,13-trinoreudesm-5-ene-7β,8α-diol (24).12 Racemosalactone A (1) was obtained as colorless crystals after crystallization from CHCl3. Its HRESIMS exhibited an [M

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RESULTS AND DISCUSSION The MeOH extract of the roots of I. racemosa was partitioned into H2O and extracted with petroleum ether, EtOAc, and nBuOH. The petroleum ether fraction was subjected to repeated column chromatography over silica gel, Sephadex LH-20, and ODS to afford five new sesquiterpene lactones, racemosalactones A−E (1−5). The 19 known compounds were identified as alantolactone (6),4a 11,13-dihydroalantolactone (7),7 iso© XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 25, 2012

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Table 1. 1H NMR Data of Compounds 1−5 (400 MHz, δ in ppm, J in Hz) 1a

position 1 2 3 4 5 6 7 8 9 11 13 14 15 a

2b

3a

1.17, 1.50, 1.41, 1.70, 1.95, 1.41, 1.70,

m m m m m m m

1.21, 1.66, 1.60, 1.84, 3.67,

2.63, m

1.12, 1.68, 1.45, 1.83, 1.57, 1.57, 2.52,

1.41, 1.86, 3.36, 4.55, 1.70, 2.03,

m dd (14.0, 12.0) ddd (12.0, 6.0, 6.0) m m dd (15.2, 5.2)

5.42, d (3.2)

5.29, s

2.97, 5.07, 1.55, 2.17,

ddd (13.2, 13.2, 3.2) ddd (13.2, 3.2, 3.2) m m m

dd (5.2, 3.2) ddd (5.2, 3.2, 2.4) m dd (15.2, 3.2)

4.53, 1.63, 2.03, 2.90,

4a

m m m m m m m

m m dd (15.2, 2.8) m

6.12, d (1.2) 5.54, d (1.2) 1.15, s

3.85, d (12.4) 3.61, d (12.4) 1.19, s

1.22, d (7.6) 1.21, s

1.03, d (7.6)

1.08, d (7.2)

1.14, d (7.6)

5a

1.81, d (12.0) 1.90, ddd (12.0, 4.0, 1.6)

5.61, d (4.4)

3.84, m 1.99, dd (12.0, 12.0) 2.68, ddd (12.0, 4.0, 1.6)

4.11, 1.73, 1.69, 1.79,

m m m m

1.64, 1.58, 2.05, 4.64, 2.38, 2.61, 2.33, 1.28,

m m m m m dd (12.8, 7.6) m d (7.6)

1.81, 1.63, 1.05, 2.29, 4.96, 1.50, 2.25,

d (12.4) ddd (13.6, 6.0, 2.0) ddd (13.6, 12.4, 12.4) m brs dd (13.2, 4.4) m

1.46, 0.79, 4.90, 4.57,

s s s s

0.91, s 0.91, d (6.4)

Recorded in CDCl3. bRecorded in methanol-d4.

Table 2. 13C NMR Data of Compounds 1−5 (100 MHz, δ in ppm) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a

1a 35.3, 28.1, 16.2, 41.0, 74.7, 36.1, 37.8, 77.4, 39.3, 35.4, 142.2, 170.8, 120.0, 23.2, 16.7,

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

2b 41.1, 26.0, 73.5, 47.0, 153.0, 117.0, 46.6, 78.3, 43.3, 33.4, 79.8, 176.4, 47.1, 29.2, 16.2,

3a

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

16.7, 32.6, 41.9, 38.6, 154.6, 117.3, 73.8, 82.0, 39.6, 33.7, 46.6, 176.1, 8.4, 28.3, 22.8,

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

4a 50.8, 67.1, 46.2, 146.1, 45.8, 21.1, 46.0, 77.0, 40.9, 34.0, 77.7, 177.0, 19.1, 18.8, 109.0,

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

5a 125.6, 63.6, 36.0, 30.7, 37.6, 36.7, 39.7, 78.9, 34.5, 144.0, 40.5, 179.9, 15.7, 19.1, 15.5,

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

Recorded in CDCl3. bRecorded in methanol-d4.

+ H − H2O]+ peak at m/z 233.1531 and established the molecular formula C15H22O3, implying an index of hydrogen deficiency of five. The IR spectrum suggested the presence of hydroxy (3439 cm−1) and α-methylene-γ-lactone functionalities (1733 and 1664 cm−1). The 1H NMR data (Table 1) showed two characteristic α-methylene-γ-lactone doublets at δH 6.12 (1H, d, J = 1.2 Hz) and 5.54 (1H, d, J = 1.2 Hz), respectively. Two methyl resonances were evident: a singlet at δH 1.15 (3H, s) and a doublet at δH 1.03 (3H, d, J = 7.6 Hz). The 13C NMR data showed 15 carbon signals (Table 2), which were assigned by a DEPT experiment as two sp2 and two sp3 quaternary carbons (including one oxygenated), three sp3 methines (including one oxygenated), one sp2 and five sp3 methylenes, and two methyls. The lactone carbonyl and olefinic resonances in the 13C NMR data (Table 2) appeared at δC 170.8 (C-12), 142.2 (C-11), and 120.0 (C-13), respectively, revealing the presence of a methylene group conjugated with a lactone carbonyl group. Therefore, the remaining index of hydrogen

deficiency identified compound 1 as a tricyclic compound. The H−1H COSY spectrum indicated the H-1/H-2/H-3/H-4/H15 and H-6/H-7/H-8/H-9 proton sequences. The HMBC cross-peaks between H-13/C-7, H-13/C-12, and H-6/C-8 indicated that a γ-lactone function was formed between C-8 and C-11. The HMBC correlations from H-14 to C-1, C-5, C9, and C-10 confirmed that Me-14 was connected to C-10, and correlations from H-15 to C-3, C-4, and C-5 confirmed that Me-15 was connected to C-4. These correlations (Figure 1) established the molecular skeleton of 1 as a eudesmane-type sesquiterpene lactone. The placement of the C-5 hydroxy group was confirmed from the HMBC correlations of the angular methyl (δH 1.15, H-14) and methyl doublet (δH 1.03, H-15) with the oxygenated quaternary carbon resonating at δC 74.7 (C-5). Thus, the molecular framework of 1 was established. The relative configuration of 1 was determined by X-ray singlecrystal diffraction studies (Figure 2). Consequently, the 1

B

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and 3268 cm−1) and γ-lactone (1748 cm−1) groups. The NMR data (Tables 1 and 2) exhibited signals typical of a eudesmanetype sesquiterpene lactone including an ester carbonyl (δC 176.4, C-12), a trisubstituted carbon−carbon double bond [δC 153.0 (C-5), 117.0 (C-6); δH 5.42 (d, J = 3.2 Hz, H-6)], and a CH2Cl group [δC 47.1 (C-13); δH 3.85, 3.61 (1H each, d, J = 12.4 Hz)]. The CH2Cl group was connected to C-11, as indicated by the HMBC correlations from H-13 (δH 3.85, 3.61) to C-12 (δC 176.4), C-11 (δC 79.8), and C-7 (δC 46.6). The H1/H-2/H-3/H-4/H-15 and H-6/H-7/H-8/H-9 proton spin systems were deduced from the 1H−1H COSY correlations. The HMBC spectrum showed the following correlations: H-1 with C-3; H-6 with C-8; H-7 with C-12; H-13 with C-11 and C-12; H-14 with C-1, C-5, and C-9; and H-15 with C-3, C-4, and C-5. These data (Figure 1) established the molecular skeleton of 2. The relative configuration of 2 was determined by X-ray crystallographic analysis (Figure 2). The presence of the chlorine atom in 2 and the value of the Flack parameter [−0.02(7)] permitted the determination of the absolute configuration of 2.13 In this way, the structure of 2 was defined as (3S,4R,7S,8R,10R,11R)-3,11-dihydroxyeudesm-5-en-12,8olide. Racemosalactone C (3) was obtained as a colorless oil and exhibited a pseudomolecular ion peak at m/z 251.1642 [M + H]+ under HRESIMS, which is consistent with a molecular formula of C15H22O3. The IR spectrum suggested the presence of hydroxy (3448 cm−1) and carbonyl (1749 cm−1) groups. The NMR data indicated that 3 was also a eudesmane-type sesquiterpene lactone similar to the known compound 7, except that it had one hydroxy group not present in 7. The presence of the C-7 hydroxy group was confirmed from the HMBC (Figure 1) correlations from H-9 (δH 2.03, dd, J = 15.2, 2.8 Hz) and H-13 (δH 1.22, d, J = 7.6 Hz) to C-7 (δC 73.8). An

Figure 1. Key 2D NMR correlations of compounds 1−5.

structure of 1 was determined as 5α-hydroxyeudesm-11(13)en-12,8β-olide. Racemosalactone B (2) was obtained as colorless, flaky crystals after crystallization from MeOH. The presence of a chloride atom was evident from a series of characteristic isotopic peaks in the ESIMS, with a ratio of 3:1 at m/z 301/303 and 318/320. Its HRESIMS showed an [M + NH4]+ signal at m/z 318.1467, suggesting a molecular formula of C15H21ClO4. The IR spectrum showed absorption bands for hydroxy (3463

Figure 2. X-ray crystal structure of compounds 1, 2, 4, and 5. C

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assays.6 It is also a major constituent of I. racemosa.5 However, previous teams have only been able to isolate 6 either at low levels of purity or after several tedious sequential purification steps.4,5,17 As previously reported, we obtained large amounts of alantolides (mixture of 6 and 8, 153 g) and only small amounts of 6 (20 mg). For this reason, we created a method that was amenable to the rapid and straightforward preparativescale isolation of 6 from alantolides. This method involves a silica gel column but does not require specialized equipment such as silica gel LC columns impregnated with AgNO3.4 As described in the Experimental Section, the alantolides were treated with selenium dioxide and tert-butyl hydroperoxide, which selectively oxidized C-3 of 8 to produce 17 while 6 remained intact (Scheme 1). This process occurred with

11,13-dihydroalantolactone skeleton was determined using the 1 H−1H COSY cross-signals for H-1/H-2/H-3/H-4/H-15, H-6/ H-7/H-8/H-9, and H-11/H-13 coupling systems and the following HMBC correlations: H-6 with C-8 and C-10; H-13 with C-7 and C-12; H-14 with C-1, C-5, C-9, and C-10; and H15 with C-3, C-4, and C-5. The relative configuration of 3 was deduced from the ROESY spectrum and biogenetic consideration. ROESY correlations (Figure 1) of H-15/H-14 and H-14/H-13 indicated that Me-13, Me-14, and Me-15 were β-oriented. The cross-peaks of H-11 and H-8 revealed that they were αoriented. On the basis of biogenetic considerations,14 the C-7 hydroxy group was predicted to be α-oriented. In this way, compound 3 was identified as 7α-hydroxyeudesm-5-en-12,8βolide. Racemosalactone D (4) was obtained as colorless crystals after crystallization from CHCl3. The HRESIMS exhibited an [M + NH4]+ peak at m/z 284.1856 and established a molecular formula of C15H22O4. The IR spectrum showed absorption bands of hydroxy (3492 and 3320 cm−1), γ-lactone (1770 cm−1), and olefinic (1649 cm−1) groups. The NMR data of 4 (Tables 1 and 2) were similar to those of 22,10 expect that a methine carbon (δC 39.9, C-11) in 22 was replaced by an oxygenated quaternary carbon (δC 77.4, C-11) in 4, as indicated by the HMBC correlations from H-13 to C-7, C-11, and C-12 (Figure 1). The connectivity was further established by means of 1H−1H COSY and HMBC experiments (Figure 1). The relative configuration of compound 4 was determined by X-ray single-crystal diffraction studies (Figure 2). Thus, the structure of compound 4 was established as 2α,11α-dihydroxyeudesm-4en-12,8β-olide. Racemosalactone E (5) was obtained as colorless crystals after crystallization from CHCl3. Its molecular formula, C15H22O3, was established using the HRESIMS spectrum (m/ z 268.1912, [M + NH4]+), implying an index of hydrogen deficiency of five. The IR absorptions were observed at 3511 and 1742 cm−1, suggesting the presence of hydroxy and carbonyl groups. The 13C NMR spectrum showed 15 carbon signals, which were assigned by a DEPT experiment to three methyl, three methylene, six methine (two oxymethine), and three quaternary carbons. The above-mentioned and the chemical shifts and splitting pattern of the resonances at δH 1.28, 0.90, and 0.92 (H3-13, H3-14, H3-15, respectively) in the 1 H NMR spectrum indicated an eremophilanolide skeleton for 5.15 The NMR data (Tables 1 and 2) showed the appearance of one trisubstituted double bond [δC 144.0 (C-10), 125.6 (C-1); δH 5.61 (d, J = 4.4 Hz, H-1)] and one ester carbonyl (δC 179.9, C-12). The presence of the three segments CH(1)−CH(2)− CH2(3)−CH (4)−CH3 (15), CH2(6)−CH (7)−CH(8)− CH2(9), and CH(7)−CH(11)−CH3(13) was inferred from the 1H−1H COSY spectrum, which showed the H-1/H-2/H-3/ H-4/H-15, H-6/H-7/H-8/H-9, and H-7/H-11/H-13 coupling systems. The HMBC correlations of H-9 with C-1 and C-5; H13 with C-7, C-11, and C-12; H-14 with C-5, C-6, and C-10; and H-15 with C-3, C-4, and C-5 established the molecular framework of 5 (Figure 1). The chemical shifts of C-1, C-2, and C-3 in 5 were similar to those of armatin B,16 suggesting that the C-2 hydroxy group was α-oriented. The structure of 5 was thus defined as 2α-hydroxyeremophil-1(10)-en-8β,12-olide. An X-ray diffraction analysis corroborated the proposed structure (Figure 2). Alantolactone (6) possesses a wide spectrum of biological activities and exhibits strong activities in those biological

Scheme 1a

a

Reagents and condition: (a) SeO2/t-BuOOH, CH2Cl2, 90%.

excellent regio- and stereoselectivity. Compounds 6 and 17 could subsequently be separated using column chromatography over silica gel. In this way, the present method showed superior isolation efficiency to previous methods. The n-hexane fraction of I. racemosa exhibits promising antiproliferative activity.5 The antiproliferative activity of some previously isolated compounds has also been investigated.10,18 All sesquiterpene lactones obtained here were screened for their antiproliferative activity against human non-small-cell lung cancer A549, hepatocellular carcinoma HepG2, and human fibrosarcoma HT1080 cells (Table 3) to identify the functional groups necessary for maintaining and increasing its activity. This may lead to the development of more effective antitumor agents. It is widely believed that α,β-unsaturated carbonyl compounds, particularly α-methylene lactones, have significant antiproliferative activity. Alkylation of biological nucleophiles by α,β-unsaturated carbonyl structures in Michael-type addition is considered the general mechanism of action.19,20 Our results were consistent with this conclusion (Table 3). The absence of an α-methylene-γ-lactone moiety diminished the antiproliferative activities of these sesquiterpene lactones, as shown by comparisons of 7 with 6, 9 with 8, 11 with 10, 13 with 12, and 14 with 15. Among these α-methylene lactones, 6 and 8 showed the most potent antiproliferative activities, with IC50 values ranging from 0.38 to 1.77 μg/mL. When 16 and 17 were compared to 8, the antiproliferative activities were reduced because of the hydroxy groups at C-5 in 16 and C-3 in 17. Oxidation of the C5−C-6 olefinic bond in 6 or the C-4−C-15 olefinic bond in 8 to an oxirane moiety, as in 12 or 14, significantly reduced antiproliferative activity. Compounds 1 and 10 showed significantly less antiproliferative activity than compound 6 because of the hydroxy group at C-5 in 1 and the C-4−C-5 olefinic bond in 10. These structure−activity relationships suggested that the relative lipophilicities of these α-methylene lactones may contribute to the activity. Inhibition of angiogenesis (antiangiogenesis) is an effective strategy in the treatment of human cancer. Thus, we evaluated the antiproliferative activity of the isolated compounds against D

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Table 3. IC50 Values for Antiproliferative Activity of Compounds 1−24 cell type (IC50, μg/mL) compound

A549

HepG2

HT1080

mitomycin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0.54 1.51 37.32 27.76 >50 38.58 0.55 24.79 0.38 >50 2.67 22.54 1.59 16.23 2.65 >50 4.27 1.47 16.22 >50 >50 24.8 >50 >50 >50

0.14 2.76 >50 >50 >50 >50 1.3 >50 1.77 >50 3.57 >50 2.73 25.3 3.78 >50 8.14 4.19 31.2 >50 >50 26.11 >50 >50 >50

0.15 1.88 >50 >50 >50 >50 0.696 >50 0.795 >50 3.87 >50 1.32 17.41 3.51 >50 3.5 2.44 28.51 >50 >50 20.1 >50 >50 >50

human umbilical vein endothelial cells (HUVECs). Compounds 6 and 8 exhibited antiproliferative activity against HUVECs, showing IC50 values of 2.4 and 2.5 μg/mL, respectively. As shown in Figure 3, they both inhibited endothelial cell tube formation at 1.0 μg/mL. These data suggest that the antiproliferative activities of compounds 6 and 8 are not more pronounced in endothelial cells than in tumor cells.

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Figure 3. Effects of compounds 6 and 8 on in vitro matrigel angiogenesis assays. Colchicine was used as a positive control.

EXPERIMENTAL SECTION

University, where a voucher specimen (accession number: 20100720) was deposited. Extraction and Isolation. The air-dried roots of I. racemosa (10.0 kg) were percolated with MeOH (3 × 20 L) at room temperature. The combined extracts were concentrated under reduced pressure, and the residue (3.2 kg) was partitioned into H2O and extracted with petroleum ether, EtOAc, and n-BuOH. The petroleum ether fraction (600 g) was subjected to flash column chromatography (CC) over silica gel (petroleum ether−EtOAc from 1:0 to 0:1, v/v) to give five major fractions (A−E). Fraction A (179 g) was recrystallized from petroleum ether to yield solid alantolides (153 g, a mixture of 6 and 8) and a mother liquor (10 g). The mother liquor was subjected to CC over silica gel (petroleum ether−EtOAc from 50:1 to 12:1, v/v) to afford three fractions, A1−A3. Fraction A1 (3.2 g) was recrystallized from petroleum ether and subjected to repeated CC over silica gel (petroleum ether− EtOAc, 20:1, v/v) to afford compounds 6 (20 mg) and 7 (12 mg). Fraction A2 (3.0 g) was subjected to CC over silica gel (petroleum ether−EtOAc, 12:1, v/v) to afford 10 (30 mg) and 11 (50 mg). Fraction A3 (2.7 g) was chromatographed over silica gel (petroleum ether−EtOAc, 12:1, v/v) and repeated CC over Sephadex LH-20 (CHCl3−MeOH, 2:1, v/v) to obtain compounds 8 (20 mg) and 9 (70 mg). Fraction B (1.5 g) was subjected to CC over silica gel (petroleum ether−EtOAc, 8:1, v/v) to give fractions B1 and B2. Fraction B1 (300 mg) was separated on a silica gel column (petroleum

General Experimental Procedures. Melting points were determined on an X-4 digital display micromelting point apparatus and are uncorrected. Optical rotations were measured on a Perkin− Elmer 341 polarimeter with 1 dm cell. IR spectra were obtained on a Nicolet NEXUS 670 FT-IR spectrometer. NMR spectra were recorded on a Bruker AVANCE III-400. Chemical shifts are given on the δ (ppm) scale using TMS as internal standard. ESI-LRMS was performed on a Finnigan LC QDECA instrument, and HRESIMS was performed on a Bruker APEX-II mass spectrometer. The X-ray diffraction data were collected on a Bruker Smart Apex CCD diffractometer, and the structures were solved by direct methods using Bruker SHELXS-97 (using graphite-monochromated Mo Kα radiation). Silica gel (200−300 mesh, Qingdao Marine Chemical Factory, China), Sephadex LH-20 (Amersham Pharmacia Biotech), and YMC Gel (ODS-A, 12 nm, S-50 μm, YMC Co., Kyoto, Japan) were used for column chromatography. Silica gel GF254 plates (10− 40 μm, Qingdao Marine Chemical Factory, China) were used for TLC. The spots on TLC were detected with 254 nm UV light and visualized by spraying with 98% H2SO4−C2H5OH (5:95, v/v) followed by heating. Plant Material. The roots of I. racemosa were collected from Gannan, Gansun Province, China, in July 2010. The plant was identified by Prof. Guo-Liang Zhang, School of Life Sciences, Lanzhou E

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C15H22O4, space group P2(1), a = 6.155(8) Å, b = 11.287(15) Å, c = 9.836(13) Å, α = 90.00°, β = 96.098(14), γ = 90.00°, V = 679.5(16) Å3, Z = 2, Dcalc =1.302 g/cm3, R1 = 0.0523, wR2 = 0.1282. Crystal Data for 5. Racemosalactone D (5) was crystallized from CHCl3 to give colorless crystals. A single crystal of dimensions 0.25 × 0.23 × 0.21 mm3 was used for X-ray measurements. Crystal data: C15H22O3, space group P2(1), a = 8.379(6) Å, b = 9.018(6) Å, c = 19.545(14) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1476.8(18) Å3, Z = 4, Dcalc = 1.207 g/cm3, R1 = 0.0455, wR2 = 0.0999. The supplementary crystallographic data for 1, 2, 4, and 5 reported in this paper have been deposited at the Cambridge Crystallographic Data Centre, under the reference numbers CCDC 897955, 897960, 897961, and 897956, respectively. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, fax: +44 1223 336033 or e-mail: data_ [email protected]. Cell Culture. Human non-small-cell lung cancer A549 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillium/streptomycin in 5% CO2 at 37 °C. Hepatocellular carcinoma HepG2 cells were cultured in Dulbecco’s modified essential medium (Gibco) with the same supplements as above. Human fibrosarcoma HT1080 cells were cultured in Alpha minimum essential medium with the same supplements as above. HUVECs were purchased from PriCells (Wuhan, China) and routinely cultured in endothelial cell medium (M&C Gene Technology, China), supplemented with 0.03 mg/mL endothelial cell growth supplement (M&C Gene Technology, China), 10% fetal bovine serum, 0.1 mg/mL heparin (M&C Gene Technology, China), and penicillium/streptomycin. HUVECs were used from the second to the sixth passages for the experiments. In Vitro Matrigel Angiogenesis Assays. According to the modified protocols,21 in vitro Matrigel angiogenesis assays were conducted in 96-well plates using HUVEC cells plated on a Matrigel layer (BD Biosciences) with 25 000 cells per well and incubated with test compounds for 22 h. Images were taken 22 h after plating. Colchicine was used as a positive control. Tube formation was quantified by measuring the total length of capillary structures using the software WCIF ImageJ. Two representative fields were counted in each experiment. Magnification was ×40. Cell Proliferation Assay. Cell proliferation was determined using CCK-8 dye (Beyotime Inst Biotech, China) according to the manufacturer’s instructions.22 Briefly, cells were seeded in a 96-well plate and allowed to adhere overnight. After incubation with compounds under evaluation for 72 h at 37 °C in 5% CO2, 10 μL of CCK-8 dyes then was added to each well, and cells were incubated at 37 °C in 5% CO2 for 2 h. Mitomycin was used as a positive control. All experiments were performed in duplicate. The absorbance was determined at 450 nm using a microplate reader (TECAN Infinite M200). Oxidation of Alantolides. A suspension of selenium dioxide (296 mg, 2.64 mmol) in DCM (10 mL) at 0 °C under Ar was added to 640 μL (5.72 mmol) of a 70% solution of tert-butyl hydroperoxide. After stirring for 30 min, a solution of alantolides (1.20 g) in 10 mL of DCM was added dropwise. The mixture was stirred at room temperature for 4 h. Aqueous NaHCO3 was added, and the mixture extracted with DCM, washed with brine, and dried over Na2SO4. The residue was purified by column chromatography on silica gel (eluent, petroleum ether−EtOAc, 3:1) to give a crude solid. Recrystallization from petroleum afforded 6 (0.60 g) and 17 (0.40 g, 90%).

ether−EtOAc, 5:1, v/v) to obtain 12 (60 mg) and 13 (15 mg). Fraction B2 (750 mg) was purified by CC over silica gel (petroleum ether−EtOAc, 5:1, v/v) to give two major subfractions, B2‑1 and B2‑2. Fraction B2‑1 (450 mg) was applied to CC over ODS (MeOH−H2O, 1:1, v/v) to afford 15 (5 mg) and 14 (40 mg). Fraction B2‑2 (150 mg) was subjected to CC over silica gel (petroleum ether−EtOAc, 4:1, v/v) to yield 1 (8 mg). Fraction C (2.1 g) was separated by CC over silica gel (petroleum ether−EtOAc, 5:1, v/v) to give two fractions, C1 and C2. Fraction C1 (900 mg) was purified by CC over silica gel (petroleum ether−EtOAc, 4:1, v/v) to give 16 (30 mg), 17 (15 mg), and 3 (20 mg). Fraction C2 (600 mg) was subjected to CC over silica gel (petroleum ether−EtOAc, 3:1, v/v) to give 18 (30 mg). Fraction D (3 g) was purified by CC over silica gel (petroleum ether−EtOAc, 5:1, v/v) to give two major fractions, D1 and D2. Fractions D1 (1.7 g) was separated on a silica gel column (petroleum ether−EtOAc, 3:1, v/v), Sephadex LH-20 (CHCl3−MeOH, 2:1, v/v), and then ODS (MeOH− H2O, 1:1, v/v) to yield 19 (50 mg), 20 (90 mg), and 5 (7 mg). Fraction D2 (1.0 g) was applied to CC over ODS (MeOH−H2O, 1:1, v/v) to afford 21 (25 mg) and 22 (43 mg). Fraction E (3 g) was separated on a silica gel column (petroleum ether−EtOAc, 2:1, v/v) to give two fractions, E1 and E2. Fraction E1 (500 mg) was purified by CC over ODS (MeOH−H2O, 1:1, v/v) to afford crude 4 (5 mg) and 23 (20 mg), and then they were recrystallized from MeOH, respectively. Fraction E2 (1.0 g) was applied to CC over ODS (MeOH−H2O, 2:3, v/v) and then Sephadex LH-20 (CHCl3−MeOH, 1:1, v/v) to afford 2 (13 mg) and 24 (5 mg). Racemosalactone A (1): colorless crystals (CHCl3); mp 125−126 °C; [α]25D +100 (c 0.1, acetone); IR (KBr) νmax 3439, 2926, 1733, 1644, 1266 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2; HRESIMS m/z 233.1531 [M + H − H2O]+ (calcd for C15H21O2, 233.1536). Racemosalactone B (2): colorless, flaky crystals (MeOH); mp 222−224 °C; [α]25D +30 (c 0.1, MeOH); IR (KBr) νmax 3463, 3268, 2928, 1748, 1645, 1224 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2; ESIMS m/z 301, 302, 303, 318, 319, 320; HRESIMS m/z 318.1467 [M + NH4]+ (calcd for C15H25ClNO4, 318.1467). Racemosalactone C (3): colorless oil; [α]25D −40 (c 0.1, acetone); IR (KBr) νmax 3448, 2929, 1749, 1651, 1198, 983 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2; HRESIMS m/z 251.1650 [M + H]+ (calcd for C15H23O3, 251.1642). Racemosalactone D (4): colorless crystals (CHCl3); mp 170−172 °C; [α]25D +100 (c 0.1, acetone); IR (KBr) νmax 3492, 3320, 2948, 1770, 1649, 1212 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2; HRESIMS m/z 284.1859 [M + NH4]+ (calcd for C15H26NO4, 284.1856). Racemosalactone E (5): colorless crystals (CHCl3); mp 80−81 °C; [α]25D −140 (c 0.1, acetone); IR (KBr) νmax 3511, 2959, 1742, 1640, 1210 cm−1; 1H (400 MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2; HRESIMS m/z 268.1912 [M + NH4]+ (calcd for C15H26NO3, 268.1907). X-ray Analysis. All measurements were collected on a Bruker Smart Apex CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The structures of 1, 2, 4, and 5 were solved by direct methods (SHELX97). Crystal Data for 1. Racemosalactone A (1) was crystallized from CHCl3 to give colorless crystals. A single crystal of dimensions 0.21 × 0.20 × 0.19 mm3 was used for X-ray measurements. Crystal data: C15H22O3, space group P2(1), a = 12.070(10) Å, b = 7.880(6) Å, c = 14.734(12) Å, α = 90.00°, β = 93.781(9)°, γ = 90.00°, V = 1398.4(19) Å3, Z = 4, Dcalc = 1.189 g/cm3, R1 = 0.0489, wR2 = 0.1068. Crystal Data for 2. Racemosalactone B (2) was crystallized from MeOH to give colorless, flaky crystals. A single crystal of dimensions 0.20 × 0.16 × 0.14 mm3 was used for X-ray measurements. Crystal data: C15H21ClO4, space group P2(1), a = 6.422(4) Å, b = 11.870(7) Å, c = 9.689(6) Å, α = 90.00°, β = 103.415(6)°, γ = 90.00°, V = 718.4(7) Å3, Z = 2, Dcalc = 1.390 g/cm3, R1 = 0.0397, wR2 = 0.0760. Crystal Data for 4. Racemosalactone D (4) was crystallized from CHCl3 to give colorless crystals. A single crystal of dimensions 0.23 × 0.22 × 0.19 mm3 was used for X-ray measurements. Crystal data:

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S Supporting Information *

1D and 2D NMR, IR, and HRESIMS spectra of compounds 1− 5 and crystal data of compounds 1, 2, 4, and 5. This material is available free of charge via the Internet at http://pubs.acs.org. F

dx.doi.org/10.1021/np300742d | J. Nat. Prod. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-931-8912592. Fax: +86-931-8912582. E-mail: [email protected]. 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 (No. 31270396) and the 111 Project of China.

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REFERENCES

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dx.doi.org/10.1021/np300742d | J. Nat. Prod. XXXX, XXX, XXX−XXX