Bioassay-Guided Isolation of Prenylated Xanthone ... - ACS Publications

Jun 22, 2016 - ABSTRACT: Four new dihydroxanthone derivatives (1−4), four new tetrahydroxanthone derivatives (5−8), two new xanthone derivatives (...
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Bioassay-Guided Isolation of Prenylated Xanthone Derivatives from the Leaves of Garcinia oligantha Yue-Xun Tang,†,‡,§ Wen-Wei Fu,†,‡,§ Rong Wu,†,‡ Hong-Sheng Tan,†,‡ Zhen-Wu Shen,† and Hong-Xi Xu*,†,‡ †

School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China Engineering Research Center of Shanghai Colleges for TCM New Drug Discovery, Shanghai 201203, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Four new dihydroxanthone derivatives (1−4), four new tetrahydroxanthone derivatives (5−8), two new xanthone derivatives (9 and 10), and two known caged tetrahydroxanthones were isolated from extracts of the leaves of Garcinia oligantha by bioassay-guided fractionation. These structures of the new compounds were elucidated by NMR and MS spectroscopic data analysis, and the absolute configurations of compounds 1 and 5−7 were determined by electronic circular dichroism and/or single-crystal X-ray diffraction analysis. Compounds 6−9 were shown to be unusual xanthone derivatives with an isopropyl group, which was confirmed by the X-ray crystallographic structure of compound 8. The inhibitory activities of these isolates against four human tumor cell lines (A549, HepG2, HT-29, and PC-3) were assayed, and compounds 1, 2, 5, 11, and 12 showed inhibitory effects on tumor cell growth, with IC50 values ranging from 2.1 to 8.6 μM. gaudichaudione H (11) and cantleyanone A (12), were isolated from an extract of the leaves of G. oligantha by bioassay-directed fractionation (Table S1, Supporting Information). Compounds 6−9 were shown to be unusual xanthone derivatives with an isopropyl group, which was confirmed from the X-ray crystallographic structure of compound 8. Oliganthin H (1) is the first dihydroxanthone derivative for which the absolute configuration has been determined through single-crystal X-ray diffraction and electronic circular dichroism (ECD) analysis, whereas oliganthic acids A−C (6−8) are rare tetrahydroxanthone derivatives with a geranyl or an isoprenyl group and a carboxylic acid group at C-8. In this report, the isolation of these compounds, the elucidation of their structures, and their inhibitory effects on the proliferation of a small panel of human cancer cells are described.

Garcinia L. (Clusiaceae) is a large genus of polygamous trees and shrubs that is distributed in tropical regions of Asia, Africa, and Polynesia. It consists of 450 species, of which 21 occur in mainland China.1 The genus is well known as a prolific source of polycyclic polyprenylated acylphloroglucinols, prenylated xanthones, and biflavonoids, which exhibit a broad array of biological activities.2 As part of a continued effort to identify potent anticancer agents from natural sources, it was found that, among extracts obtained from various parts of 14 Garcinia plants, the 95% EtOH extract of the leaves of Garcinia oligantha Merr. (Guttiferae) exhibited the most potent cytotoxic activity against 10 human cancer cell lines.3 G. oligantha, a shrub 1−3 m tall, is distributed mainly in 200−1200 m dense forests in the Guangdong and Hainan provinces of the People’s Republic of China. In folkloric medicine, this plant is purported to alleviate inflammation, cool internal heat, and afford detoxification of the body.4 To date, only a few compounds with cytotoxic properties from this plant have been reported, and no phytochemical research on the leaves of G. oligantha has been conducted.4 In this work, four new dihydroxanthone derivatives (1−4), four new tetrahydroxanthone derivatives (5−8), two new xanthone derivatives (9, 10), and two known caged xanthones, © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The petroleum ether- and EtOAc-soluble portions of the 90% EtOH extract of G. oblongifolia leaves were purified separately by column chromatography over MCI gel, silica gel, reversed-phase Received: February 13, 2016

A

DOI: 10.1021/acs.jnatprod.6b00137 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

C18 silica gel, and Sephadex LH-20 and by preparative HPLC, yielding 12 compounds, including 10 new (1−10) and two known caged xanthones (Figure S2, Supporting Information). The known caged xanthones were identified as gaudichaudione H (11)5 and cantleyanone A (12)6 by comparison of their spectroscopic data with published values, and the absolute configurations of 11 and 12 were determined for the first time as 5R, 7R, 10aS, and 22S. Oliganthin H (1) was obtained as a yellow, amorphous powder. Its molecular formula was determined to be C33H38O7 by HRESIMS at m/z 545.2546 [M − H]−, suggesting the presence of 15 degrees of unsaturation. The IR spectrum showed the presence of hydroxy groups (3290 cm−1), aromatic rings (1610 cm−1), and a xanthone carbonyl group (1651 cm−1). The 1 H and 13C NMR data of 1 (Table 1) indicated the occurrence of seven methyls, four methylenes, six olefinic methines, and 16 quaternary carbons (two carbonylic, three oxygenated and two nonoxygenated aromatic, three oxygenated and four nonoxygenated olefinic, and two sp3 quaternary), including those for a prenyl group [δH 1.38 (3H, s, H3-19), 1.41 (3H, s, H3-20), 3.26 (1H, m, H-16a), 3.89 (1H, m, H-16b), and 5.18 (1H, t, J = 6.4 Hz, H-17); δC 38.5 (C-16), 120.0 (C-17), 134.9 (C-18), 26.1(C-19), and 18.0 (C-20)], a 3,7-dimethyl-2,6-octadienyl group [δH 1.59 (3H, s, H3-28), 1.63 (3H, s, H3-29), 1.67 (3H, s, H3-30), 1.81, 1.90 (both 2H, m, H2-24 and H2-25, respectively), 3.26 (1H, m, H-21a), 3.89 (1H, m, H-21b), 4.94 (1H, t, J = 8.5 Hz, H-26), and 5.22 (1H, m, H-22); δC 38.2 (C-21), 120.2 (C22), 138.9 (C-23), 40.4 (C-24), 27.5 (C-25), 124.9 (C-26), 131.6 (C-27), 26.1 (C-28), 18.6 (C-29), and 16.9 (C-30)], and a dimethyl-2H-pyrano group [δH 7.07 (1H, d, J = 10.0 Hz, H-11), 5.57 (1H, d, J = 10.0 Hz, H-12), 1.40 (6H, H3-14 and H3-15); δC

115.7 (C-11), 127.8 (C-12), 78.9 (C-13), and 28.6 (C-14 and C15)]. On the basis of this evidence and analysis of its 2D NMR spectrum, compound 1 was deduced to be based on a xanthenedione skeleton bearing a prenyl, a geranyl, and a dimethyl-2H-pyrano group.4,7 The prenyl and 3,7-dimethyl-2,6-octadienyl groups were located at C-8 (δC 55.2) like those in garcinianone A,7 according to the HMBC correlations from H2-16 to C-7, C-8, C-8a, and C21 and from H2-21 to C-7, C-8, C-8a, and C-16. The dimethyl2H-pyrano group was shown to be attached at C-3 and C-4 on the basis of the HMBC correlations between the cis-coupled H11 and H-12 and C-4, between H-11 and C-3 and C-4a, and between H-12 and C-13, C-14, and C-15. The remaining aromatic proton was indicated to be H-2 (δH 6.56), from HMBC correlations with C-1, C-3, C-4, and C-9a. On considering the signals for δC 150.7 (C-5) and 141.2 (C-6) and the molecular formula of 1, two hydroxy groups could be located at C-5 and C6, respectively. The substitution pattern and the assigned planar structure of 1 were confirmed by complete 1H−1H COSY and HMBC spectroscopic analysis. Selected key correlations in the observed NMR spectrum are shown in Figure 1a. The NOESY correlations of H2-21/H3-30 and H-22/H2-24 of 1 suggested the 22E configuration for the 3,7-dimethyl-2,6octadienyl group (Figure 1a). Since there is a single chiral center (C-8) in the molecule, there were only two possible isomers, (8R)-1 and (8S)-1. The absolute configuration of 1 was determined by comparison of the experimentally measured ECD curve with the TDDFT-predicted curves. The results showed that the calculated ECD curve of (8R)-1 was consistent with the experimental ECD spectrum of 1 (Figure 1b). Moreover, an X-ray diffraction experiment with a suitable crystal B

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Table 1. 1H and 13C NMR Spectroscopic Data (600 and 150 MHz) for Compounds 1−4a 1b

a c

position

δC, type

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11a 11b 12 13 14 15 16a 16b 17 18 19 20 21a 21b 22 23 24 25 26 27 28 29 30 OH-1 OH-5 OH-7

163.4 C 100.9 CH 160.0 C 102.0 C 151.9 C 150.7 C 141.2 C 193.8 C 55.2 C 119.4 C 180.6 C 106.1 C 157.7 C 115.7 CH 127.8 CH 78.9 C 28.6 CH3 28.6 CH3 38.5 CH2 120.0 CH 134.9 C 26.1 CH3 18.0 CH3 38.2 CH2 120.2 CH 138.9 C 40.4 CH2 27.5 CH2 124.9 CH 131.6 C 26.1 CH3 18.6 CH3 16.9 CH3

2b δH (J in Hz) 6.56, s

7.07, d (10.0) 5.57, d (10.0) 1.40, s 1.40, s 3.26, m 3.89, m 5.18, t (6.4) 1.38, s 1.41, s 3.26, m 3.89, m 5.22, m 1.81, m 1.90, m 4.94, t (8.5)

δC, type

3c δH (J in Hz)

163.4 C 100.9 CH 160.0 C 102.0 C 151.8 C d 150.4 C 141.2 C 193.8 C 55.1 C 119.3 C 180.6 C 106.1 C 157.7 C 115.7 CH

6.56, s

7.02, d (10.0)

127.8 CH 78.9 C 28.6 CH3 28.6 CH3 38.4 CH2

5.55, d (10.0) 1.39, s 1.39, s 3.25, m 3.88, m 5.18, t (7.1)

118.5 CH 135.0 C 26.2 CH3 18.6 CH3 38.4 CH2

1.38, s 1.62, s 3.25, m 3.88, m 5.18, t (7.1)

118.5 CH 135.0 C 26.2 CH3 18.6 CH3

1.38, s 1.62, s

1.59, s 1.63, s 1.67, s 14.02, s

14.01, s

δC, type 163.1 C 99.5 CH 161.9 C 93.7 CH 156.8 C 122.8 C 149.1 C 200.1 C 55.4 C 117.2 C 179.5 C 105.3 C 159.7 C 37.8 CH2 118.1 CH 135.2 C 25.8 CH3 18.1 CH3 37.8 CH2 118.1 CH 135.2 C 25.8 CH3 18.1 CH3 23.6 CH2 119.2 CH 134.7 C 25.9 CH3 18.2 CH3

4c δH (J in Hz) 6.29, d (2.1) 6.37, d (2.1)

2.76, m 3.41, m 4.58, t (7.5) 1.46, s 1.46, s 2.76, m 3.41, m 4.58, t (7.5) 1.46, s 1.46, s 3.41, m 5.13, t (7.3) 1.68, s 1.81, s

δC, type 160.9 C 99.8 CH 160.6 C 104.6 C 153.9 C 56.0 C 201.5 C 151.9 C 108.8 CH 116.6 C 179.8 C 105.3 C 159.2 C 21.9 CH2 121.2 CH 135.5 C 26.0 CH3 18.1 CH3 38.0 CH3 117.9 CH 135.5 C 25.9 CH3 18.1 CH3 38.0 CH2 117.9 CH 135.5 C 25.9 CH3 18.1 CH3

13.29, s 7.03, s

δH (J in Hz) 6.31, s

6.51, s

3.51, d (7.0) 5.30, t (6.7) 1.78, s 1.87, s 2.80, m 3.43, m 4.64, t (7.3) 1.49, s 1.48, s 2.80, m 3.43, m 4.64, t (7.3) 1.49, s 1.48, s

13.15, s 6.99, s

1

b

1

Assignments are based on DEPT, HSQC, HMBC, and H− HCOSY experiments. Chemical shifts are given in ppm. Measured in pyridine-d5. Measured in CDCl3. dOverlapped with the solvent peak. The data in parentheses are coupling constants (J) in Hz.

Figure 1. (a) Key correlations observed in the NMR spectra, (b) calculated and experimental ECD curves, and (c) ORTEP diagram of compound 1.

was conducted by Cu Kα radiation with a Flack parameter of 0.00(19) (Figure 1c), and the absolute configuration of 1 was established as 8R (Figure 1), consistent with the absolute configuration determined by ECD calculation. Thus, the structure of 1 was assigned as shown.

Oliganthin I (2) was also isolated as a yellow, amorphous powder, and its molecular formula was assigned as C28H30O7 based on HRESIMS data. Both the UV (λmax 235, 280 nm) and IR [νmax 3402 (hydroxy group), 1743 (unconjugated carbonyl group), 1655 (a xanthone carbonyl group), 1618 (aromatic ring) C

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Table 2. 13C NMR Data (150 MHz) for Compounds 5−10a position

5b

6b

7c

8b

9c

10c

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

164.9 C 98.1 CH 163.3 C 101.9 C 155.2 C 84.3 C 201.7 C 84.9 C 134.6 C 132.0 C 178.7 C 101.1 C 89.7 C 115.6 CH 127.4 CH 78.5 C 28.5 CH3 28.5 CH3 28.7 CH2 117.6 CH 135.6 C 25.7 CH3 17.0 CH3 29.9 CH2 49.8 CH 83.7 C 30.6 CH3 29.2 CH3

160.0 C 100.4 CH 161.9 C 105.7 C 154.5 C 69.3 CH 45.9 CH 34.7 CH2 49.2 C 116.1 C 182.9 C 105.0 C 166.5 C 21.8 CH2 121.3 CH 135.2 C 25.8 CH3 18.1 CH3 34.4 CH2 117.8 CH 140.3 C 40.1 CH2 26.7 CH2 124.0 CH 131.7 C 25.9 CH3 17.8 CH3 16.6 CH3 175.5 C 144.0 C 113.7 CH2 19.9 CH3

159.1 C 98.9 CH 163.7 C 106.4 C 153.5 C 181.5 C 126.0 C 36.5 CH2 47.8 C 127.2 C 181.7 C 104.3 C 152.3 C 21.1 CH2 122.1 CH 130.9 C 25.5 CH3 17.7 CH3 33.5 CH2 120.4 CH 137.2 C 39.9 CH2 25.3 CH2 124.0 CH 130.7 C 26.0 CH3 17.4 CH3 15.8 CH3 175.3 C 150.3 C 23.3 CH3 23.1 CH3

161.5 C 100.5 CH 160.7 C 101.7 C 151.3 C 181.5 C 125.7 C 36.5 CH2 48.1 C 127.7 C 182.3 C 106.3 C 153.3 C 115.0 CH 127.3 CH 78.7 C 28.5 CH3 28.4 CH3 34.1 CH2 120.3 CH 134.9 C 26.0 CH3 17.8 CH3 179.6 C 152.9 C 24.1 CH3 24.0 CH3

162.5 C 98.4 CH 159.7 C 100.6 C 150.7 C 141.1 C 142.2 C 123.1 C 132.6 C 115.8 C 182.8 C 103.4 C 146.5 C 115.5 CH 126.9 CH 78.1 C 27.9 CH3 27.9 CH3 33.0 CH2 123.8 CH 131.0 C 25.6 CH3 17.9 CH3 27.1 CH 22.1 CH3 22.1 CH3

162.5 C 98.0 CH 158.9 C 100.2 C 150.4 C 130.7 C 142.1 C 141.2 C 118.7 C 109.3 C 182.2 C 102.9 C 142.4 C 115.4 CH 126.8 CH 77.9 C 27.8 CH3 27.8 CH3 24.7 CH2 124.3 CH 129.5 C 25.6 CH3 18.0 CH3

a

Assignments are based on DEPT, HSQC, and HMBC experiments. Chemical shifts are given in ppm. bMeasured in CDCl3. cMeasured in DMSOd6.

cm−1] spectra of oliganthin I showed absorption characteristics similar to those of oliganthin A,4 indicating that they have similar carbon skeletons. The 1H NMR data (Table 1) of 2 showed the characteristic signals for a dimethyl-2H-pyrano group [δH 7.02 (1H, d, J = 10.0 Hz, H-11), 5.55 (1H, d, J = 10.0 Hz, H-12), 1.39 (6H, H3-14 and H3-15)], a gem-bis(3-methylbut-2-enyl) group [δH 5.18 (2H, t, J = 7.1 Hz, H-17 and H-22), 3.88 (2H, m, H-16b and H-21b), 3.25 (2H, m, H-16a, and H-21a), 1.62 (6H, s, H3-20 and H3-25), and 1.38 (6H, s, H3-19 and H3-24)], an aromatic singlet proton [δH 6.56 (1H, s, H-2)], and a hydrogen-bonded hydroxy group [δH 14.01 (1H, s), OH-1]. The 13C NMR and DEPT spectra of 2 indicated six CH3, two CH2, five CH, and 15 C signals, including those for a dimethyl-2H-pyrano group, a gem-bis(3-methylbut-2-enyl) group, a quaternary carbon (δC 55.1, C-8), a tertiary aromatic carbon [δC 100.9 (C-2)], two nonoxygenated quaternary aromatic carbons, a nonoxygenated quaternary olefinic carbon [δC 102.0 (C-4), 106.1 (C-9a), and 119.3 (C-8a)], three oxygenated aromatic and three oxygenated olefinic carbons [δC 141.2 (C-6), 151.8 (C-4a), 150.4 (C-5), 157.7 (C-10a), 160.0 (C-3), and 163.4 (C-1)], and two carbonyl carbons [δC 180.6 (C-9) and 193.8 (C-7)]. In contrast, the NMR data of 2 were quite similar to those of oliganthin A except at C-2 and C-4. In 2, a C5 group could be located at C-4 instead of at C2, forming a dimethyl-2H-pyranoxanthone skeleton at C-3. The HMBC correlations from H-11 to the oxygenated aromatic

carbons C-3 and C-4a, from H-12 to the aromatic carbon C-4, and from H-12 to the oxygenated quaternary carbon C-13 and the tertiary methyl carbons C-14 and C-15 confirmed the above deduction. The structure of 2 was confirmed by DEPT, HSQC, 1 H−1H COSY, and HMBC experiments. Selected key HMBC and 1H−1H COSY correlations are shown (Figure S3, Supporting Information). On the basis of these results, the structure of compound 2 was established as shown. Oliganthin L (3) gave a molecular formula of C28H32O6 by HRESIMS at m/z 463.2128 [M − H]−. The NMR data of 3 were similar to those of 2, indicating that these two compounds have similar carbon skeletons. The different carbon chemical shifts for C-4 and C-5 indicated that the structure of 3 differs from that of 2 with respect to the substituents attached at C-4 and C-5. The NMR data indicated that a prenyl group [δH 3.41 (2H, m, H-21), 5.13 (1H, t, J = 7.3 Hz, H-22), 1.68 (3H, H3-24), and 1.81 (3H, H3-25); δC 23.6 (C-21), 119.2 (C-22), 134.7 (C-23), 25.9 (C24), and 18.2 (C-25)] is present at C-5 in 3 instead of a dimethyl2H-pyrano group as in 2. Two meta-substituted aromatic protons [δH 6.29 (1H, d, J = 2.1 Hz, H-2), 6.37 (1H, d, J = 2.1 Hz, H-4); δC 99.5 (C-2), 93.7 (C-4)] in the 1H and 13C NMR data (Table 1), together with the HMBC correlations from H-21 to C-5, C-6, and C-10a and from −OH at C-6 to C-6 and C-7, confirmed the above deduction (Figure S3, Supporting Information). ConD

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Table 3. 1H NMR Data (600 MHz) for Compounds 5−10a 5b

position 2 5 6 7

6.03, s

8 11

7.50, d (1.2) 6.63, d (10.1)

12 14 15 16a 16b 17 19 20 21a 21b 22 23 24 25 28 29 OH-1 OMe-7

5.56, d (10.1) 1.45, s 1.45, s 2.57, m 4.46, m 1.39, s 1.09, s 2.36 br d (12.7) 1.63, dd (13.0, 9.8) 2.57, d (9.7) 1.69, s 1.32, s

12.59, s 3.64, s

6b

7c

8b

9c

10c

6.35, s 4.52, d (10.3) 2.69, m 1.59, m 2.31, dd (14.0, 2.2)

6.33, s

6.15, s

6.18, s

2.94, s

2.97, s

7.02, s

3.46, dd (15.7, 7.2) 3.53, dd (15.7, 6.8) 4.82, t (7.2) 1.73, s 1.81, s 2.80, dd (14.6, 7.8) 2.94, dd (14.5, 7.1) 4.82, t (7.2) 1.93, m 1.93, m 4.95, tq (5.6, 4.0, 2.0)

3.35, s

6.84, d (10.0)

7.30, d (10.0)

7.15, d (10.0)

5.22, t (7.5) 1.59, s 1.77, s 2.60, dd (14.3, 7.4) 2.94, m 5.02, t (7.8) 1.77, m 1.77, m 4.87, br s

5.56, d (10.0) 1.44, s 1.44, s 2.71, d (7.5)

5.73, d (10.1) 1.43, s 1.43, s 3.88, d (6.9)

5.73, d (10.0) 1.44, s 1.44, s 3.95, d (6.3)

5.01, br s 1.51, s 1.34, s

5.32, t (6.9) 1.67, s 1.71, s 3.43, m

5.16, t (6.0) 1.60, s 1.75, s

1.52, s 1.42, s 1.34, s 2.14, s 1.91, s

2.22, s 1.89, s

1.62, s 1.51, s 1.59, s 5.00, d (15.4) 1.84, s 11.95, s

6.13, s

1.22, s 1.21, s

a Assignments are based on DEPT, HSQC, and HMBC experiments. Chemical shifts are given in ppm. bMeasured in CDCl3. cMeasured in DMSOd6. The data in parentheses are coupling constants (J) in Hz.

Figure 2. (a) Key HMBC and 1H−1H COSY correlations, (b) key NOESY correlations observed in the NMR spectra, and (c) calculated and experimental ECD curves of compound 5.

from H-8 (δH 6.51) to C-6 (δC 201.5), C-8a (δC 116.6), C-9 (δC 179.8), and C-10a (δC 159.2) and from H2-16 and H2-21 (δH 2.80 and 3.41) to C-5 (δC 56.0) and C-6 (δC 201.5) indicated the presence of a gem-bis(3-methylbut-2-enyl) group at C-5 instead of at C-8 as in 2 and 3. Furthermore, on the basis of analysis of the HMBC correlations from H-11 (δH 3.51) to C-3 (δC 160.6), C-4 (δC 104.6), and C-4a (δC 153.9), a prenyl group [δH 3.51 (2H, d, J = 7.0 Hz, H2-11), 5.30 (1H, t, J = 6.7 Hz, H-12), 1.78 (3H, s, H314), and 1.87 (3H, s, H3-15); δC 21.9 (C-11), 121.2 (C-12), 135.5 (C-13), 26.0 (C-14), and 18.1 (C-15)] could be located at the C-4 position. The structure of 4 was confirmed by 1H−1H COSY and HMBC experiments (Figure S3, Supporting Information). Thus, the structure of 4 (oliganthin K) was established as shown. Oliganthone B (5) was isolated as a yellow, amorphous powder with a molecular formula of C29H32O7 as determined by HRESIMS (m/z 515.2052 [M + Na]+, calcd for 515.2046). Both the UV (λmax 206, 232, 274, 360 nm) and IR [νmax 1745

sequently, the structure of compound 3 was established as shown. The HRESIMS of oliganthin K (4) showed an ion peak at m/z 463.2125 [M − H]−, yielding the molecular formula C28H32O6. The IR spectrum exhibited bands for hydroxy groups (νmax 3396 cm−1) and a xanthone carbonyl group (νmax 1647 cm−1). Furthermore, the NMR data of 4 were similar to those of 1−3, indicating that 4 is also a xanthenedione derivative. The 1H and 13 C NMR data of 4 (Table 1) showed the presence of a hydrogen-bonded hydroxy group [δH 13.15 (1H, s, OH-1)] and two singlet protons [δH 6.31 (1H, s, H-2) and 6.51 (1H, s, H-8), δC 99.8 (C-2) and 108.8 (C-8)]. The presence of a set of signals [δH 1.48 (6H, s, H3-20 and H3-25), 1.49 (6H, s, H3-19 and H324), 2.80 (2H, m, H-16a and H-21a), 3.41 (2H, m, H-16b and H21b), and 4.64 (2H, t, J = 7.3 Hz, H-17, H-19); δC 38.0 (C-16, C21), 117.9 (C-17, C-22), and 135.5 (C-18, C-23)] was typical of a gem-bis(3-methylbut-2-enyl) group linked to an sp3 carbon atom at δC 56.0, like those found in 2 and 3. The HMBC correlations E

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Figure 3. (a) Key HMBC and TOCSY correlations, (b) key NOESY correlations observed in the NMR spectra, and (c) calculated and experimental ECD curves of compound 6.

14.0, 2.2 Hz, H-7b)], a nonoxygenated methine proton [δH 2.69 (1H, m, H-6)], an oxygenated methine proton [δH 4.52 (1H, d, J = 10.1 Hz, H-5)], an aromatic proton [δH 6.35 (1H, s, H-2)], and a chelated phenolic proton [δH 11.95 (1H, s, OH-1)]. The 13C NMR and DEPT spectra of 6 indicated the presence of six CH3, six CH2, six CH, and 14 C signals, including those for a prenyl group, a 3,7-dimethyl-2,6-octadienyl group, an isopropenyl group, a methylene carbon (δC 21.8, C-7), two methine carbons [δC 45.9 (C-6) and 69.3 (C-5)], a quaternary carbon (δC 49.2, C8), a tertiary aromatic carbon (δC 98.9, C-2), two nonoxygenated quaternary aromatic carbons [δC 105.0 (C-9a) and 105.7 (C-4)], three oxygenated aromatic and one oxygenated olefinic carbon [δC 154.5 (C-4a), 160.0 (C-1), 161.9 (C-3), and 166.5 (C-10a)], and two carbonyl carbons [δC 175.5 (C-26) and 182.9 (C-9)]. A contiguous spin system comprising H-5, H-6, and H2-7 was present in the TOCSY spectrum, which revealed, together with the correlations from the HMBC spectrum (Figure 3a), the connectivity from C-5 to C-8. These findings suggested that compound 6 is a 5,6,7,8-tetrahydroxanthone derivative. The 3,7-dimethyl-2,6-octadienyl group and carboxylic acid of compound 6 were both located at C-8 (δC 49.2), according to the HMBC correlations between H2-16 and C-8 and C-26 and between H2-7 and C-16 and C-26. The isopropenyl group was proven to be attached at C-6 by the HMBC correlations between C-6 and H3-29 and H2-28 and those from H-5, H-6, and H2-7 to C-27. HMBC correlations were also observed from −OH-1 to C1, C-2, and C-9a and from H-11 to C-3, C-4, and C-4a, indicating attachments of the isoprenyl group to C-4. The substitution pattern and the assigned planar structure of 6 were further confirmed by DEPT, HSQC, TOCSY, and HMBC NMR experiments. The key TOCSY and HMBC correlations for 6 are shown in Figure 3a. The relative configuration of 6 was determined by a NOESY experiment and by analysis of the 1H NMR spectroscopic coupling constants. In the NOESY spectrum, NOE correlations between H-5 and H-7a and between H-6 and H-7b were observed (Figure 3b). This, in combination with the 3J values (10.1 Hz) observed for H-5, indicated the presence of a half-chair form of a tetrahydrobenzene ring with both H-5 and H-6 in axial positions. In addition, the NOESY correlations of H2-16/H3-25 and H-17/H2-19 suggested the 17E configuration for the 3,7dimethyl-2,6-octadienyl group. There were three chiral centers evident (C-5, C-6, and C-8), so the four possible isomers, (5R,6R,8R)-6, (5R,6R,8S)-6, (5S,6S,8R)-6, and (5S,6S,8S)-6, were considered. The absolute configuration of 6 was

(unconjugated carbonyl group), 1622 (ortho-hydroxy chelated carbonyl group) cm−1] spectra showed absorption characteristics of a caged xanthone.6,8 Except for the substituent at C-4, the 13C and 1H NMR data (Tables 2 and 3) were also comparable to those of gaudichaudione H. The NMR data indicated a dimethyl2H-pyrano group [δH 6.63 (1H, d, J = 10.1 Hz, H-11), 5.56 (1H, d, J = 10.1 Hz, H-12), and 1.45 (6H, s, H3-14 and H3-15); δC C 115.6 (C-11), 127.4 (C-12), 78.5 (C-13), 28.5 (C-14, and C15)] at C-4 in 5 instead of a prenyl group as in gaudichaudione H. The HMBC correlations observed from H-11 to C-3 (δC 163.3), C-4 (δC 101.9), and C-4a (δC 155.2), together with the molecular formula of C29H32O7, confirmed the above deduction. The structure of 5 was confirmed by 1H−1H COSY and HMBC experiments (Figure 2a). The relative configuration of 5 was assessed by analysis of the NOESY data. NOESY correlations were observed between H-8 and H-21b, H-25 and 21a, H-24 and H-11, and H-24 and H-22 (Figure 2b). The NOESY experiment supported the same relative stereochemistry for the tricyclic core of 5 as previously reported for other caged xanthone derivatives.6,9 In addition, the ECD spectrum (Figure 2c) exhibited Cotton effects (CEs) similar to those observed for (−)-morellic acid.8 The two compounds both displayed sequential negative and positive CEs near 360, 290, 246, and 215 nm, indicating them to have the same absolute configuration at C-5, C-7, C-10a, and C-22. On the basis of the NOESY correlations and the CEs in the ECD spectrum, the absolute configuration of 5 was determined to be 5R, 7R, 10aS, and 22S. This assignment was confirmed unambiguously by its theoretically calculated ECD spectrum (Figure 2c). Thus, the structure of 5 (oliganthone B) was established as shown. Oliganthic acid A (6) was obtained as a yellow, amorphous powder. A molecular formula of C32H40O7 was suggested by HRESIMS (m/z 535.2708 [M − H]−, calcd for 535.2696). The IR spectrum exhibited bands for hydroxy groups (3359) and aromatic rings (1589 cm−1). The 1H NMR data (Table 3) of 6 showed characteristic signals for a prenyl group [δH 1.73 (3H, s, H3-14), 1.81 (3H, s, H3-15), 3.46 (1H, dd, J = 15.7, 7.2 Hz, H11a), 3.53 (1H, dd, J = 15.7, 6.8 Hz, H-11b), and 4.82 (1H, t, J = 7.2 Hz, H-12)], a 3,7-dimethyl-2,6-octadienyl group [δH 1.51 (3H, s, H3-24), 1.59 (3H, s, H3-23), 1.59 (3H, s, H3-25), 1.93 (4H, m, H2-19 and H2-20), 2.80 (1H, dd, J = 14.6, 7.8 Hz, H16a), 2.94 (1H, dd, J = 14.5, 7.1 Hz, H-16b), 4.82 (1H, t, J = 7.2 Hz, H-17), and 4.95 (1H, m, H-21)], an isopropenyl group [δH 1.84 (3H, s, H-28) and 5.00 (2H, d, J = 15.4 Hz, H2-29)], two methylene protons [δH 1.59 (1H, m, H-7a) and 2.31 (1H, dd, J = F

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Figure 4. (a) Key HMBC and TOCSY correlations, (b) key NOESY correlations observed in the NMR spectra, and (c) calculated and experimental ECD curves of compound 7.

HMBC correlation of H2-16 with C-7, C-8, C-8a, and C-21, those of H-11 with C-3, C-4, and C-4a, and that of H-12 with C-4 (Figure S4, Supporting Information), together with the molecular formula of C27H28O7, confirmed the above deduction. Although there is a single chiral center (C-8), 8 was found to be a racemic mixture that displayed no Cotton effects in the ECD spectrum (Figure S79, Supporting Information). Moreover, an X-ray diffraction experiment with the mixed crystals of compound 8 was conducted by Cu Kα radiation, and this compound was shown to be an unusual xanthone derivative with an isopropyl group, which was confirmed from the X-ray crystallographic structure of (8S)-8 (Figure S6, Supporting Information). Thus, the structure of (±)-oliganthic acid C was deduced as shown. The 13C and 1H NMR assignments observed for this compound are shown in Tables 2 and 3. The molecular formula of oliganthaxanthone A (9) was established as C26H28O5 by HRESIMS. Its UV (258 and 336 nm) and IR (3442 and 1649 cm−1) absorptions were typical of a xanthone derivative.6 The 1H NMR data (Table 3) of 9 showed signals characteristic of a dimethyl-2H-pyrano group [δH 7.30 (1H, d, J = 10.0 Hz, H-11), 5.73 (1H, d, J = 10.1 Hz, H-12), 1.43 (s, 6H, H3-14, and H3-15); δC 115.5 (C-11), 126.9 (C-12), 78.1 (C-13), and 27.9 (C-14, and C-15)], a prenyl group [δH 3.88 (2H, d, J = 6.9 Hz, H2-16), 5.32 (1H, t, J = 6.9 Hz, H-17), 1.67 (3H, s, H3-19), and 1.71 (3H, s, H3-20); δC 33.0 (C-16), 123.8 (C-17), 131.0 (C-18), 25.6 (C-19), and 17.9 (C-20)], and an isopropyl group [δH 3.43 (1H, m, H-21), 1.22 (3H, s, H3-22), and 1.21 (3H, s, H3-23); δC 27.1(C-21) and 22.1 (C-22 and C-23)]. The foregoing data indicated that 9 is a xanthone derivative with a prenyl group, a dimethyl-2H-pyrano unit, and an isopropyl structural fragment. The prenyl group was located at C-8 according to the HMBC correlations between H2-16 and C-7, C8, and C-8a, and the isopropyl group was located at C-6 according to the HMBC correlations between H-21 and C-5, C6, and C-7. The dimethyl-2H-pyrano group was proved to be attached at C-4 by the HMBC correlations from the cis-coupled H-11 and H-12 to C-4 and those between H-12 and the oxygenated quaternary carbon C-13 and the tertiary methyl carbons C-14 and C-15. The remaining aromatic protons were indicated to be δH 6.18 (1H, H-2) and 7.02 (1H, s, H-7) by the HMBC correlations (Figure S4, Supporting Information). The structure assigned for 9 was confirmed by DEPT, HSQC, TOCSY, and HMBC NMR experiments. Thus, the structure of 9 was designated as shown. Oliganthaxanthone B (10) was obtained as a yellow, amorphous powder with a [M − H]− ion at m/z 409.1287 in

determined by comparison of the experimentally measured ECD curve with the TDDFT-predicted curves. The results showed that the calculated ECD curve of (5R,6R,8S)-6 was consistent with the experimental ECD spectrum of 6 (Figure 3c). Thus, the absolute configuration of 6 was determined as shown in Figure 3. Oliganthic acid B (7) was also obtained as a yellow, amorphous powder. The HRESIMS showed an ion peak at m/z 533.2537 [M + H]−, giving the molecular formula C32H38O7. The NMR data of 7 were similar to those of 6, indicating that the two compounds have similar carbon skeletons. However, the different 13C NMR chemical shifts observed for C-5 and C-6 indicated that the structure of 7 differs from that of 6 with respect to the substituents attached at these two positions. The NMR data indicated the presence of a 2-methylpropene group [δH 1.91 (3H, s, H3-29) and 2.14 (3H, s, H3-28); δC 120.6 (C-6), 150.3 (C-27), 23.3 (C-28), and 23.1 (C-29)] in 7 instead of an isopropenyl group at C-6. The 13C NMR data of 7 showed C-5 as an oxygenated quaternary carbon, and considering the molecular formula and the signal at δC 181.5 (C-5), a carbonyl group could be located at C-5. The HMBC correlations of H3-28 and H3-29 with C-6 and C-27 and of H2-7 with C-5, C-6, and C-27 confirmed this deduction. The assigned planar structure of 7 was confirmed by DEPT, HSQC, TOCSY, and HMBC NMR experiments. The key TOCSY and HMBC correlations for 7 are shown in Figure 4a. In addition, NOESY correlations of H2-16/ H3-25 and H-17/H2-19 suggested the 17E configuration for the 3,7-dimethyl-2,6-octadienyl group, as in compounds 1 and 6 (Figure 4b). There were only two possible isomers, (8R)-7 and (8S)-7, due to the single chiral center (C-8). The absolute configuration of 7 was determined as (8S)-7 by comparison of the experimentally measured ECD curve with the TDDFTpredicted curves (Figure 4c), thus establishing the assignment of the absolute configuration of 7 as depicted. The HRESIMS of (±)-oliganthic acid C (8) showed an ion peak at m/z 465.1918 [M + H]+, giving the molecular formula C27H28O7. In contrast, the NMR data of 8 were quite similar to those of 7 except for the substituents at C-4 and C-8. The NMR data indicated that a prenyl group [δH 2.71 (2H, d, J = 5.1 Hz, H16), 5.01 (1H, m, H-17), 1.51 (3H, H3-19), and 1.34 (3H, H320); δC 34.1 (C-16), 120.3 (C-17), 134.9 (C-18), 26.0 (C-19), and 17.8 (C-20)] was present at C-8 in 8 instead of a 3,7dimethyl-2,6-octadienyl group as in 7 and that a dimethyl-2Hpyrano group [δH 6.84 (1H, d, J = 10.0 Hz, H-11), 5.56 (1H, d, J = 10.0 Hz, H-12), and 1.44 (6H, s, H3-14 and H3-15); δC 115.0 (C-11), 127.3 (C-12), 78.7 (C-13), 28.5 (C-14), and 28.4 (C15)] was present at C-4 instead of an isoprenyl group as in 7. The G

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Table 4. Cytotoxicity of Isolated Compounds against Cancer Cell Linesa compound

A549

HepG2

HT-29

PC-3

HL-7702c

1 2 5 11 12 paclitaxelb

5.0 ± 0.32 5.5 ± 0.47 3.9 ± 0.86 3.0 ± 0.49 2.9 ± 0.42 0.08 ± 0.013

>10 6.2 ± 0.39 4.5 ± 1.20 2.8 ± 0.32 2.9 ± 0.38 0.20 ± 0.043

6.4 ± 0.32 4.1 ± 0.18 4.8 ± 0.99 2.2 ± 0.30 2.3 ± 0.64 0.02 ± 0.020

5.9 ± 0.42 3.2 ± 0.29 4.6 ± 0.78 2.1 ± 0.41 2.3 ± 0.61 0.03 ± 0.014

>10 6.4 ± 0.60 8.6 ± 0.75 2.5 ± 0.34 3.1 ± 0.30 0.06 ± 0.018

a Results are expressed as mean IC50 values in μM, and compounds 3, 4, and 6−10 are inactive in these human cancer cell lines (IC50 > 10 μM). Values represent the mean ± SD of three independent experiments. bPositive control. cHuman normal hepatic cells.

HPLC was performed on a Waters 2535 Series machine equipped with an Xbridge C18 column (4.6 × 250 mm, 5 μm), and preparative HPLC was performed on a preparative Xbridge Prep C18 OBD column (19 × 250 mm, 5 μm). Column chromatography was performed on CHP20P MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Japan), silica gel (200−300 mesh, Qingdao Haiyang Chemical Co., Ltd.), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden), and reversed-phase C18 silica gel (50 μm, YMC, Kyoto, Japan). Analytical and preparative TLC was performed on precoated GF254 plates (0.25 or 0.5 mm thickness, Qingdao Haiyang Chemical Co. Ltd.). Detection was performed by spraying the plates with 10% sulfuric acid followed by heating. Plant Material. Leaves of G. oligantha were collected in August 2013, at Bawangling in Changjiang Li Autonomous County of Hainan Province, People’s Republic of China. The plant was identified by Dr. Rong-Jing Zhang, Department of Plant Sciences, the College of Life Sciences, South China Agricultural University, and a specimen (SHTYX-201309) was deposited at the Engineering Research Center of Shanghai Colleges for TCM New Drug Discovery. Extraction and Isolation. Air-dried and powdered leaves of the plant (2.4 kg) were extracted successively with petroleum ether (5 × 12 L, 2 days each time) and 90% EtOH (v/v, 5 × 12 L, 2 days each time) at room temperature. The combined extracts were evaporated to dryness under vacuum to obtain a petroleum ether-soluble portion (fraction A, 30 g) and a 90% ethanol-soluble portion (460 g). The 90% ethanolsoluble portion was suspended in H2O (2 L) and extracted with EtOAc (3 × 2 L) to yield an EtOAc-soluble portion (fraction B1, 286 g) and a H2O-soluble portion (fraction B2). Fractions A and B1 were shown to have cytotoxic activity against the PC-3 cancer cell line (Table S1, Supporting Information). Fraction A (30.0 g) was chromatographed on an MCI gel column, eluting with EtOH−H2O (30:70, 60:40, 90:10, and 95:5, successively), to yield five fractions (A1−A5). Fraction A1 (5.3 g) was separated on Sephadex LH-20 in CHCl3−MeOH (1:1), yielding four subfractions (A1.1−A1.4). Subfractions A1.2 (3.7 g) and A1.4 (37.7 mg) were further purified on Sephadex LH-20 and eluted with CHCl3−MeOH (1:1) and MeOH to obtain compounds 11 (0.5 g) and 10 (11.0 mg), respectively. Fraction A2 (4.0 g) was chromatographed over a reversed-phase C18 silica gel column (4 × 60 cm) eluted with a gradient (30:70 to 100:0) of MeOH−H2O, yielding subfractions A2.1−A2.13. Subfraction A2.11 (0.7 g) was further purified by Sephadex LH-20 with MeOH to yield compound 2 (200 mg). Fraction A3 (3.8 g) was separated on a reversedphase C18 silica gel column (4 × 60 cm) eluted with a gradient of MeOH−H2O (30:70 to 100:0), yielding 10 subfractions (A3.1−A3.10). In return, subfractions A3.5 (40 mg) and A3.6 (52 mg) were further purified on Sephadex LH-20 in MeOH to afford compounds 5 (2.6 mg) and 12 (3.0 mg), respectively. Fraction B1 (286.0 g) was subjected to silica gel column chromatography using a gradient of dichloromethane−MeOH (100:0 to 50:50, v/v), and this yielded five fractions (B1.1−B1.5) based on their TLC profiles. Fraction B1.1 (24.0 g) was chromatographed separately on an MCI gel column eluted with EtOH−H2O (30:70, 60:40, 90:10, and 95:5) to afford 11 subfractions (B1.1.1−B1.1.11) using TLC. Fraction B1.1.3 (2.5 g) was separated initially using a reversed-phase C18 silica gel column eluted with MeOH−H2O (30:70 to 100:0) in a step gradient, yielding six subfractions (B1.1.3.1−B1.1.3.6). Subfraction B1.1.3.1 (70 mg) was further purified by preparative HPLC (MeCN−

the HRESIMS, consistent with a molecular formula of C23H22O7 (calcd for C23H21O7, m/z 409.1287). The UV spectrum showed absorption bands consistent with the presence of aromatic rings and conjugated carbonyl groups. The IR spectrum exhibited bands for hydroxy groups and a xanthone carbonyl group. Furthermore, the NMR data of 10 were similar to those of 9, indicating that the two compounds are based on the same carbon skeleton but with differences at C-6 and C7. Compound 10 did not exhibit signals for an isopropyl group and showed only one aromatic proton signal, in contrast to compound 9. Considering the signal for δC 142.1 (C-6) and 141.2 (C-7) and the molecular formula of 10, two hydroxy groups could be located at C-6 and C7, respectively. The assigned structure of 10 was confirmed by DEPT, HSQC, TOCSY, and HMBC NMR experiments (Figure S4, Supporting Information). Thus, the structure of 10 was designated as shown. Although the 1H and 13C NMR signals of gaudichaudione H (11) and cantleyanone (12) were assigned by previous investigators,5,6 the issue of defining their absolute configurations has not been addressed. These two compounds and oliganthone B (5) exhibited well-matched ECD spectra (Figure S5, Supporting Information), and they all displayed negative and positive Cotton effects near 360 and 290 nm, respectively. Since the absolute configuration of oliganthone B (5) was defined unambiguously by a combination of experimental and theoretically calculated ECD spectra, the absolute configurations of 11 and 12 were all determined to be 5R, 7R, 10aS, and 22S, based on their similar NMR and ECD data. In the present investigation, all isolates obtained were evaluated for their cytotoxic activities against the A549 (lung adenocarcinoma), HepG2 (hepatoma carcinoma), HT-29 (colorectal adenocarcinoma), PC3 (prostate cancer), and HL7702 (human normal liver) cell lines using the MTT method with paclitaxel as the positive control. As shown in Table 4, compounds 1, 2, 5, 11, and 12 exhibited cytotoxic activity against three or four of the human cancer cell lines with IC50 values close to or less than 10 μM. Among these cytotoxic compounds, only compound 1 did not show cytotoxicity to normal hepatic HL7702 cells, indicating some selective toxicity toward the cancer cells.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using an Autopol VI 90079 polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra were recorded on a Varian Cary 50 spectrophotometer. ECD spectra were measured on a Chirascan v.4.2.17 spectropolarimeter. IR spectra were obtained on a Nicolet 6700 spectrophotometer. The NMR spectra were recorded on a Bruker Avance-600 NMR spectrometer and calibrated based on the solvent peak used. ESIMS and HRESIQTOFMS experiments were performed on an Agilent 1100 Series MSD trap mass spectrometer and an Agilent 6520 ESI-Q-TOF spectrometer, respectively. Analytical H

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(±)-Oliganthic Acid C (8): yellow, amorphous powder; [α]20D +16.7 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 239 (4.41), 286 (4.29) nm; IR (KBr) νmax 3423, 2974, 2924, 1712, 1653, 1579, 1485, 1433, 1375, 1279, 1176, 1115, 768 cm−1; 1H and 13C NMR (CDCl3) data, Tables 2 and 3; HRESIMS m/z 465.1918 [M + H]+ (calcd for C27H29O7, m/z 465.1913). Oliganthaxthanone A (9): yellow, amorphous powder; UV (MeOH) λmax (log ε) 258 (4.36), 336 (3.86) nm; IR (KBr) νmax 3431, 2918, 1718, 1618 cm−1; 1H and 13C NMR (DMSO-d6) data, Tables 2 and 3; HRESIMS m/z 463.2128 [M − H]− (calcd for C28H31O6, m/z 463.2121). Oliganthaxthanone B (10): yellow, amorphous powder; UV (MeOH) λmax (log ε) 203 (4.53), 211 (4.52), 267 (4.49), 336 (4.00) nm; IR (KBr) νmax 3419, 2960, 2920, 2850, 1649 cm−1; 1H and 13C NMR (DMSO-d6) data, Tables 2 and 3; HRESIMS m/z 409.1287 [M − H]− (calcd for C29H33O7, m/z 409.1287). X-ray Crystallographic Analysis of Compounds 1 and (8S)-8. The crystallographic data were collected on a Bruker APEX II CCD-based diffractometer using graphite-monochromated Cu Kα radiation at 296(2) K for 1 and 173(2) K for (8S)-8. Using Olex2,10 the structure was solved with the ShelXS11 structure solution program using direct methods and refined with the ShelXL11 refinement package using CGLS minimization. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were placed in calculated positions employing a riding model. Crystallographic data of compound 1 and (8S)-8 have been deposited in the Cambridge Crystallographic Data Centre as CCDC 1473506 and 1480592. The data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif (or from the CCDC, 12 Union Road, Cambridge CB21EZ, U.K.; fax: +44-1223-336-033; e-mail: [email protected]). Crystal Data for Compound 1. C33H38O7; fw = 546.63; triclinic space group P1 (no. 1); unit cell dimensions a = 9.9020(3) Å, b = 12.4670(4) Å, c = 14.0279(4) Å, V = 1490.86(8) Å3, α = 63.8855(18)°, β = 78.520(2)°, γ = 74.356(2)°; μ(Cu Kα) = 0.687 mm−1; Z = 2; Dcalc = 1.218 g/cm3; 19 844 reflections measured (8.072° ≤ 2θ ≤ 139.008°), 8188 unique (Rint = 0.0474, Rsigma = 0.0737), which were used in all calculations. The final R1 was 0.0543 (I > 2σ(I)) and wR2 was 0.1587 (all data). Crystal Data for Compound (8S)-8. C27H28O7; fw = 464.51; monoclinic space group C2/c (no. 15); unit cell dimensions a = 34.17(2) Å, b = 9.894(4) Å, c = 14.754(8) Å, V = 4776(5) Å3, β = 106.76(4)°; Z = 8; Dcalc = 1.302 g/cm3; μ(Cu Kα) = 0.783 mm−1; 4363 reflections measured (5.402° ≤ 2θ ≤ 136.866°), 4363 unique (Rsigma = 0.0304), which were used in all calculations. The final R1 was 0.0997 (I > 2σ(I)) and wR2 was 0.2860 (all data). Cytotoxicity Assays. The cytotoxic activities of all isolates were evaluated by an MTT assay using A549 cells (lung adenocarcinoma), HepG2 cells (hepatocellular carcinoma), HT-29 cells (colorectal adenocarcinoma), PC-3 cells (prostate cancer), and HL-7702 cells (a normal human liver cell line). Paclitaxel was used as a positive control. The detailed methodology for the cytotoxicity assay has been described in a previous report.12

H2O, 70:30, 20 mL/min), yielding compounds 7 (30.2 mg) and 6 (6.6 mg). Fraction B1.1.5 (2.7 g) was subjected to silica gel column chromatography, using a gradient of petroleum ether−EtOAc (10:1 to 0:1, v/v), to afford three fractions (B1.1.5.1−B1.1.5.3) based on TLC. Subfraction B1.1.5.3 (0.8 g) was chromatographed on a Sephadex LH20 column in MeOH and further purified by preparative HPLC (MeCN−H2O, 70:30, 20 mL/min), yielding compound 8 (8.0 mg). Fraction B1.1.8 (2.2 g) was also subjected to silica gel column chromatography using a gradient of petroleum ether−EtOAc (10:1 to 0:1, v/v), which yielded three fractions (B1.1.8.1−B1.1.8.7) based on the TLC profiles. Subfraction B1.1.8.2 (200.0 mg) was further purified by preparative HPLC with MeCN−H2O (75:25) to give compounds 4 (10.0 mg) and 3 (9.4 mg). Subfraction B1.1.8.6 (1 g) was subjected to silica gel column chromatography in a gradient of petroleum ether− EtOAc (8:1 to 1:1, v/v). From this, subfraction B1.1.8.6.7 (44.3 mg) was purified on Sephadex LH-20 in MeOH to yield compound 9 (3.0 mg). Fraction B1.1.10 (4.8 g) was separated by Sephadex LH-20 in CHCl3− MeOH (1:1), yielding four subfractions (B1.1.10.1−B1.1.10.4). Then, subfraction B1.1.10.2 (4 g) was subjected to reversed-phase C18 silica gel column chromatography and eluted in a step gradient of MeOH−H2O (30:70 to 100:0) to give 10 subfractions. Finally, subfraction B1.1.10.2.6 (0.6 g) was purified on Sephadex LH-20 in MeOH, yielding compound 1 (500 mg). Oliganthin H (1): yellow, amorphous powder; [α]20D +19.2 (c 0.055, MeOH); UV (MeOH) λmax (log ε) 204 (4.74), 235 (4.49), 282 (4.32), 407 (3.82) nm; ECD (c 8.79 × 10−4 M, MeOH) λmax nm (Δε) 203 (+3.91), 255 (−1.75), 246 (+0.64), 289 (+1.56); IR (KBr) νmax 3290, 2970, 2918, 1651, 1610, 1576, 1481, 1458, 1433, 1385, 1278, 1149, 1113, 968, 833, 733 cm−1; 1H and 13C NMR (pyridine-d5) data, Table 1; HRESIMS m/z 545.2546 [M − H]− (calcd for C33H37O7, 545.2539). Oliganthin I (2): yellow, amorphous powder; UV (MeOH) λmax (log ε) 235 (4.60), 280 (4.43) nm; IR (KBr) νmax 3402, 3213, 2964, 2922, 1676, 1655, 1618, 1676, 1655, 1618, 1576, 1485, 1429, 1346, 1313, 1265, 1157, 1113 cm−1; 1H and 13C NMR (pyridine-d5) data, Table 1; HRESIMS m/z 477.1902 [M − H]− (calcd for C29H34O7, m/z 477.1913). Oliganthin J (3): yellow, amorphous powder; UV (MeOH) λmax (log ε) 203 (4.70), 290 (4.21) nm; IR (KBr) νmax 3431, 2918, 1718, 1618, 1385, 1149, 1099, 839 cm−1; 1H and 13C NMR (CDCl3) data, Table 1; HRESIMS m/z 419.1850 [M − H]− (calcd for C26H27O5, m/z 419.1858). Oliganthin K (4): yellow, amorphous powder; UV (MeOH) λmax (log ε) 302 (4.02), 358 (3.77) nm; IR (KBr) νmax 3396, 3193, 2920, 2850, 1647, 1468, 1421, 1379, 1119, 650 cm−1; 1H and 13C NMR (CDCl3) data, Table 1; ESITOFMS m/z 463.2125 [M − H]− (calcd for C28H31O6, m/z 463.2121). Oliganthone B (5): yellow, amorphous powder; [α]20D −253 (c 0.025, MeOH); UV (MeOH) λmax (log ε) 207 (4.51), 232 (4.43), 274 (4.39), 360 (4.05) nm; ECD (c 1.22 × 10−3 M, MeOH) λmax nm (Δε) 200 (+22.87), 222 (+30.98), 244 (−12.99), 289 (+25.22), 319 (+16.28), 360 (−46.76); IR (KBr) νmax 3444, 2920, 2850, 1745, 1622, 1541, 1385, 1259, 1078, 800 cm−1; 1H and 13C NMR (CDCl3) data, Tables 2 and 3; HRESIMS m/z 515.2052 [M + Na]+ (calcd for C29H32O7Na, m/z 515.2046). Oliganthic Acid A (6): yellow, amorphous powder; [α]20D +44.2 (c 0.057, MeOH); UV (MeOH) λmax (log ε) 202 (4.64), 257 (4.44), 301 (3.87) nm; ECD (c 1.05 × 10−3 M, MeOH) λmax nm (Δε) 203 (+55.78), 222 (−23.72), 264 (−27.61), 313 (+2.37); IR (KBr) νmax 3359, 2829, 1712, 1589, 1412, 1142, 1113, 1063 cm−1; 1H and 13C NMR (CDCl3) data, Tables 2 and 3; HRESIMS m/z 535.2708 [M − H]− (calcd for C32H39O7, 535.2696). Oliganthic Acid B (7): yellow, amorphous powder; [α]20D −82.5 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 203 (4.47), 206 (4.45), 218 (4.46), 285 (4.15), 328 (3.78) nm; ECD (c 9.36 × 10−4 M, MeOH) λmax nm (Δε) 211 (+23.69), 240 (+2.51), 286 (−15.94), 334 (+4.22); IR (KBr) νmax 3386, 2966, 2916, 1711, 1649, 1616, 1585, 1506, 1429, 1375, 1279, 1184, 1074, 812 cm−1; 1H and 13C NMR (DMSO-d6) data, Tables 2 and 3; HRESIMS m/z 533.2537 [M − H]− (calcd for C32H37O7, 533.2539).

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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.jnatprod.6b00137. Computational details for compounds 1 and 5−7 and the HRESIMS, UV, IR, ECD, and NMR spectra of compounds 1−10 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax (H.-X. Xu): +86-21-51323089. E-mail: [email protected]. I

DOI: 10.1021/acs.jnatprod.6b00137 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by the National Natural Science Foundation of China (No. 81303266), Natural Science and Technology Foundation for Distinguished Young Scholars of Shanghai (13ZR1462000), Chen Guang Foundation of Shanghai Ministry of Education (13CG46 ), Science Foundation for the Excellent Youth Scholars of Ministry of Education of Shanghai (ZZszy13058), and the Foundation of Shanghai University of Traditional Chinese Medicine (2012JW05 and 2013JW08). We are grateful to Dr. H. Zhang of Shanghai University of Traditional Chinese Medicine for measuring HRESIQTOFMS spectra.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00137 J. Nat. Prod. XXXX, XXX, XXX−XXX