Cytotoxic Germacrane-Type Sesquiterpene Lactones from the Whole

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Cytotoxic Germacrane-Type Sesquiterpene Lactones from the Whole Plant of Carpesium lipskyi Nan-Lin Zhu,†,‡ Chunping Tang,†,§ Chenghui Xu,∥ Chang-Qiang Ke,†,§ Ge Lin,§ Janar Jenis,⊥ Sheng Yao,†,§ Hongchun Liu,*,∥ and Yang Ye*,†,‡,§,¶,#

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†

Natural Products Chemistry Department and ∥Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, People’s Republic of China § SIMM-CUHK Joint Research Laboratory for Promoting Globalization of Traditional Chinese Medicines, Shanghai 201203, People’s Republic of China ⊥ Research Center for Medicinal Plants, Al-Farabi Kazakh National University, Al-Farabi Avenue 71, 050040 Almaty, Republic of Kazakhstan ¶ Collaborative Innovation Center of Southwest Ethnic Medicine, Guangxi Normal University, Guiling 541001, People’s Republic of China # School of Life Science and Technology, ShanghaiTech University, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Ten new sesquiterpene lactones, carlipsines A−J (1−10), and 12 known analogues (11−22) were isolated from the whole plant of Carpesium lipskyi. Their structures were elucidated by using 1D and 2D NMR and HRESIMS analyses, and their absolute configurations were confirmed by X-ray diffraction studies. All compounds were identified as germacranolides with diverse substructural features. Compounds 1−4 are 2,5-hemiacetal-linked germacranolides. Compounds 5 and 6 possess a 1,2-epoxy moiety. Compounds 7 and 8 represent unusual 1,5-hemiacetal-linked germacranolides. Compounds 9 and 10 contain a tetrahydrofuran unit with the oxygen atom bridging C-1 and C-8. Compounds 6, 7, 8, 19, 20, 21, and 22 showed cytotoxicity against HL-60 and A-549 cell lines with IC50 values ranging from 2.8 to 10.3 μM.

S

ities. Owing to the presence of a plethora of stereogenic centers and a multitude of oxygenated functionalities, the assignment of absolute configuration of germacranolides represents a daunting task. In our continuing efforts on searching for natural products with novel structures and potent bioactivities, Carpesium lipskyi Winkl., belonging to the family Compositae, was found to be rich in germacranolides using an in-house LC-MS method. A systematic investigation of C. lipskyi was conducted, resulting in the isolation of 22 germacranolides including 10 new analogues. Their structures were determined by extensive

esquiterpenoid lactones exhibit an array of potent bioactivities including cytotoxic1−5 and anticancer6,7 effects and have caught the attention of a number of researchers in recent years. Artemisinin derivatives, well known for their therapeutic effects on malaria, were found to display anticancer efficacy.8 Arglabin, a guaianolide sesquiterpenoid, has been clinically used for the treatment of breast, lung, and liver cancers in Kazakhstan.9 Parthenolide, a germacranolide isolated from Tanacetum parthenium, exhibited promising antitumor efficacy.10 In the past five years, germacranolides had been reported in more than 250 publications. Germacranolides exhibited cytotoxic,2,4,5,7 antiprotozoal,11,12 antiproliferative,13 antibacterial,14 anti-inflammatory,15−19 and antiplasmodial20 bioactiv© XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 28, 2018

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DOI: 10.1021/acs.jnatprod.8b01004 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

H2-2′/H-3′, H-3′/H3-4′, H-3′/H3-5′, H2-2″/H-3′′, H-3′′/H34″, and H-3′′/H3-5″, together with the HMBC correlations from H2-2′ (δH 2.35, 2.13) to C-1′ (δC 172.6) and from H2-2″ (δH 2.27, 2.19) to C-1″ (δC 172.2), indicated the presence of two 3-methylbutyryloxy groups (Figure 1). Apart from these two substituents, the remaining 15 carbon resonances were indicative of a sesquiterpenoid skeleton. The HMBC correlations from H2-13 (δH 6.36, 5.70) to C-11 (δC 133.3), C-12 (δC 168.5), and C-7 (δC 44.8) and from H-7 (δH 3.03) and H-8 (δH 5.18) to C-12 established a lactone moiety (Figure 1). HMBC correlations from H-2 (δH 4.67) to C-5 (δC 106.0) indicated the presence of an epoxy moiety. The spin systems inferred from the 1H−1H COSY spectrum (Figure 1), H3-15/H-4/H2-3/H-2/H2-1 and H-6/H-7/H-8, together with the HMBC correlations from H3-14 (δH 1.24) to C-1 (δC 44.0), C-9 (δC 78.2), and C-10 (δC 72.2), from H-9 (δH 4.60) and H-7 (δH 3.03) to C-8 (δC 77.3), and from H-6 (δH 5.13) and H3-15 (δH 1.12) to C-5 (δC 106.0), revealed that compound 1 contained a 10-membered ring with Me-14 located at C-10 and Me-15 at C-4. Comparing the NMR data with those of the known divaricin B (17)21 suggested that they shared the same skeleton except for the two acyl groups. The location of the 3-methylbutyryloxy groups at C-6 and C-9 were confirmed by the HMBC correlations between H-9 (δH 4.60) and C-1′ (δC 172.6) and between H-6 (δH 5.13) and C-1″ (δC 172.2) (Figure 1). The relative configuration was inferred from the NOESY experiment (Figure 2). The NOESY correlations of H-9/H-7 and H-9/Me-14 suggested that these protons were cofacial and β-oriented, while the cross-peaks of H-8/H-6, H-2/H-3α, and H3-15/H-3α indicated that H-2, H-6, H-8, and Me-15 were on the other face and α-oriented. The absolute configuration was established by a single-crystal X-ray crystallographic diffraction experiment with Cu Kα radiation (CCDC 1875786, Figure 4). Therefore, the structure of carlipsine A (1) was defined as (2S,4R,5R,6R,7R,8S,9R,10S)-2,5-epoxy-5,10-dihydroxy-6,9di(3-methylbutyryloxy)germacran-8,12-olide. Carlipsine B (2) was obtained as colorless, monoclinic crystals. The HRESIMS and 13C NMR data gave a molecular formula of C24H36O9 with seven indices of hydrogen deficiency. The IR data indicated the presence of hydroxy (3479 cm−1) and carbonyl groups (1723 and 1779 cm−1). Analysis of 1D and 2D NMR data (Table 1) suggested high similarities between the structures of 2 and 1 except for the differences observed for the two substituents. Three spin systems, H3-3′/H-2′/H3-4′, H2-2″/H-3′′, and H3-4″/H-3′′/ H3-5″ observed in the 1H−1H COSY spectrum, together with the HMBC correlations from H-2′ (δH 2.67) to C-1′ (δC 176.5) and from H2-2″ (δH 2.18) to C-1″ (δC 172.2), indicated the presence of a 3-methylbutyryloxy and an isobutyloxy group. The 3-methylbutyryloxy and isobutyloxy groups were located at C-6 and C-9, respectively, based on the HMBC correlations from H-6 (δH 5.13) to C-1″ and from H-9 (δH 4.62) to C-1′. The relative configuration of 2 was established by a NOESY experiment. The NOESY cross-peaks of H-7/H-9 and H-9/Me-14 indicated the β-orientation of these protons and methyl groups, whereas the cross-peak of H-6/H-8 and H2/Me-15 indicated that these protons and methyl groups occupied the α-face. The absolute configuration of 2 was defined as the same as 1 by the Cu Kα radiation X-ray crystallographic data analysis (CCDC 1875787, Figure 4). Therefore, the structure of carlipsine B (2) was defined as

analyses of 1D and 2D NMR and HRESIMS data, as well as Xray diffraction studies. All isolates were evaluated for cytotoxicity against human cancer cell lines HL-60 and A549. Herein, the isolation and structure elucidation of new compounds 1−10 and their cytotoxic activities are described. Chart 1

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RESULTS AND DISCUSSION A 95% ethanol extract of the whole plant of C. lipskyi was suspended in water and partitioned with petroleum ether, CH2Cl2, and EtOAc, successively. The CH2Cl2 extract was subjected to repeated column chromatography over silica gel, MCI gel, and Sephadex LH-20 and semipreparative and preparative HPLC, affording 10 new sesquiterpenoids named carlipsines A−J (1−10, Chart 1), along with 12 known analogues (11−22, Figure S1, Supporting Information). Carlipsine A (1) was obtained as colorless monoclinic crystals. Its HR-ESIMS peak at m/z 483.2600 ([M + H]+, calcd for 483.2594) and the 13C NMR data suggested a molecular formula of C25H38O9 with seven indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3479 cm−1) and carbonyl groups at 1734 and 1729 cm−1. The 1H NMR data (Table 1) showed two exocyclic olefinic protons (δH 6.36, 5.70), four oxygenated methines (δH 5.18, 5.13, 4.67, 4.60), and six methyl groups (δH 1.24, 1.12, 0.98, 0.95, 0.92, 0.90). The 13C NMR data (Table 1) displayed 25 resonances ascribed to six methyls, five methylenes, eight methines (four oxygenated), a quaternary carbon, three ester carbonyls, an oxygenated tertiary carbon, and a dioxygenated secondary carbon. The 1H−1H COSY correlations between B


Journal of Natural Products Table 1. 1H and

13

Article

C NMR Data for Compounds 1−5 in CDCl3 (δ in ppm, J in Hz)

1

2

3

4

δH, mult (J in Hz)

δH, mult (J in Hz)

δH, mult (J in Hz)

δH, mult (J in Hz)

5

no.

δC

1

44.0, CH2

1.76, m

44.1, CH2

1.80, m

44.1, CH2

1.79, m

44.0, CH2

1.83, m

60.2, CH

2 3

74.1, CH 37.3, CH2

74.2, CH 37.4, CH2

36.9, CH

36.9, CH

4.69, m α 2.02, m β 1.75, m 2.51, m

53.2, C 33.8, CH2

36.9, CH

4.69, m α 2.02, m β 1.76, m 2.50, m

74.0, CH 37.4, CH2

36.8, CH

4.69, m α 2.01, m β 1.75, m 2.50, m

74.2, CH 37.4, CH2

4

4.67, m α 1.99, m β 1.73, m 2.49, m

5 6 7

106.0, C 75.7, CH 44.8, CH

5.13, d (10.9) 3.03, m

106.1, C 75.8, CH 44.9, CH

5.13, d (10.9) 3.03, m

106.1, C 75.8, CH 44.8, CH

5.12, d (10.9) 3.05, m

106.0, C 75.8, CH 44.8, CH

5.11, d (10.9) 3.06, m

8

77.3, CH

9 10 11 12 13

78.2, CH 72.2, C 133.3, C 168.5, C 127.3, CH2

14 15 1′ 2′

30.0, CH3 14.7, CH3 172.6, C 43.1, CH2

3′

25.5, CH

4′ 5′ 1″ 2″

22.5, CH3 22.6, CH3 172.2, C 43.1, CH2

3″ 4″ 5″

25.3, CH 22.5, CH3 22.5, CH3

5.18, dd (10.0, 1.6) 4.60, d (10.0)

6.36, d (1.7) 5.70, d (1.7) 1.24, s 1.12, d (6.5) 2.35, m 2.13, m 2.09, m 0.98, d (7.4) 0.90, d (7.4) 2.27, 2.19, 2.09, 0.92, 0.95,

m m m d (6.6) d (6.6)

δC

77.3, CH 78.1, CH 72.3, C 133.3, C 168.4, C 127.4, CH2

5.20, dd (10.0, 1.4) 4.62, d (10.0)

6.36, d (1.3) 5.70, d (1.3) 1.25, s 1.14, d (6.5)

δC

77.2, CH

5.19, dd (10.1, 1.6) 4.61, d (10.1)

78.0, CH 72.3, C 133.3, C 168.4, C 127.3, CH2

31.0, CH3 14.7, CH3 176.5, C 34.3, CH

2.67, m

31.0, CH3 14.7, CH3 176.0, C 41.3, CH

19.2, CH3

1.24, d (6.5)

26.7, CH2

19.2, CH3

1.21, d (6.5)

172.2, C 43.1, CH2

2.18, m

11.7, CH3 16.7, CH3 172.2, C 43.1, CH2

25.4, CH 22.5, CH3 22.6, CH3

2.09, m 0.94, d (7.0) 0.92, d (7.0)

25.4, CH 22.5, CH3 22.6, CH3

6.36, d (2.3) 5.70, d (2.3) 1.25, s 1.13, d (6.6)

δC

77.3, CH 77.9, CH 72.1, C 133.0, C 168.3, C 127.2, CH2

6.37, d (1.9) 5.71, d (1.9) 1.25, s 1.16, d (6.6)

38.7, CH 208.9, C 77.0, CH 46.2, CH 72.1, CH 75.4, CH 70.9, C 132.7, C 168.0, C 127.4, CH2

δH, mult (J in Hz) 2.70, m − 3.31, m 1.58, m 2.35, m 3.16, qd (6.8, 2.0) 4.86, d (10.7) 3.25, dq (10.7, 1.3) 4.61, dd (8.4, 1.3) 4.78, d (8.4)

6.44, d (1.4) 5.89, d (1.4) 1.32, s 1.21, d (6.8)

2.67, m

23.9, CH3 19.0, CH3 176.7, C 34.1, CH

2.68, m

18.9, CH3

1.21, d (6.9)

18.5, CH3

1.18, d (6.8)

19.0, CH3

1.24, d (6.9)

19.1, CH3

1.25, d (6.8)

2.19, m

176.1, C 41.0, CH

2.35, m

166.0, C 125.6, C

2.09, m 0.94, d (6.6) 0.95, d (6.6)

26.8, CH2 11.6, CH3 16.8, CH3

1.45, m 0.89, t (7.2) 1.09, d (7.2)

143.2, CH 6.25, m 16.1, CH3 2.01, m 20.6, CH3 1.95, m

2.47, m 2.00, 1.50, 0.96, 1.18,

m m m m

30.8, CH3 14.5, CH3 176.4, C 34.1, CH

5.20, dd, (10.0, 1.7) 4.61, d (10.0)

δC

Carlipsine C (3) had a molecular formula of C25H38O9 as defined by the HRESIMS and 13C NMR data (Table 1). A comparison of the NMR data of 3 with those of 1 revealed their isomeric nature. The 13C NMR data (Table 1) indicated the resonances of a 3-methylbutyryloxy group at δC 172.2, 43.1, 25.4, 22.5, and 22.6. The presence of a 2-methylbutyryloxy group was deduced from the HMBC correlations from H-2′ (δH 2.47) to C-1′ (δC 176.0), C-3′ (δC 26.7), C-4′ (δC 11.7), and C-5′ (δC 16.7). The HMBC correlations from H-9 (δH 4.61) to C-1′ and from H-6 (δH 5.12) to C-1″ suggested the attachment of the 3-methylbutyryloxy group to C-6, and the 2-methylbutyryloxy group to C-9. The NOESY correlations of H-9/H-7 and H-9/Me-14 indicated that H-7, H-9, and Me-14 were cofacial and β-oriented, while those of H-8/H-6, H-2/H-3α, and Me-15/H-3α indicated that H-2, H-6, H-8, and Me-15 were α-oriented. Therefore, based on biosynthetic considerations,4 the structure and absolute configuration of carlipsine C (3) may be assumed as (2S,4R,5R,6R,7R,8S,9R,10S)-2,5-epoxy-5,10-dihydroxy-9-(2methylbutyryloxy)-6-(3-methylbutyryloxy)germacran-8,12olide. The HRESIMS data of carlipsine D (4) suggested a molecular formula of C24H36O9. A comparison of the NMR

Figure 1. Key 1H−1H COSY and HMBC correlations of compounds 1, 5, 7, and 9.

(2S,4R,5R,6R,7R,8S,9R,10S)-2,5-epoxy-5,10-dihydroxy-6-(3methylbutyryloxy)-9-isobutyloxygermacran-8,12-olide. C



Article

Figure 3. NOESY correlations of compounds 7 and 9.

from HMBC correlations of H2-13 (δH 6.44 and 5.89) to C-7 (δC 46.2) and C-12 (δC 168.0) and from H-7 (δH 3.25) to C12. HMBC correlations from H-6 (δH 4.86) to C-1″ (δC 166.0) and from H-9 (δH 4.78) to C-1′ (δC 176.7) revealed that the angeloyloxy and isobutyloxy groups were attached to C-6 and C-9, respectively. The NOESY correlations (Figure 2) of H-9/H-7 and H-9/Me-14 suggested the β-orientations of H9, H-7, and Me-14 and α-orientations of H-8 and H-6. In addition, the NOESY correlations of H-3″/Me-5′′ suggested a Z-configuration of the Δ(2″,3′′) olefinic bond. The absolute configuration was confirmed by Cu Kα X-ray crystallographic data analysis (CCDC 1875785, Figure 5). Therefore, the structure of carlipsine E (5) was defined as (1S,2R,4R,6S,7R,8S,9R)-1,2-epoxy-6-angeloyloxy-10-hydroxy-9-isobutyloxy5-oxogermacran-8,12-olide. The HRESIMS data of compound 6 suggested a molecular formula of C24H34O9. The NMR data (Table 2) of 6 were highly similar to those of 5 except for the signals of the C-6 substituent. Compound 6 had a 3-methylbutyryloxy group instead of the angeloyloxy group in 5 attached to C-6, which was supported by the characteristic NMR signals, as well as the HMBC correlations from H-6 (δH 4.81) to C-1″ (δC 171.7). The NOESY correlations of H-9/H-7 and H-9/Me-14 suggested the β-orientations of H-9, H-7, and Me-14 and αorientations of H-8 and H-6. Therefore, based on biosynthetic considerations,4 the structure and absolute configuration of carlipsine F (6) was assumed as (1S,2R,4R,6S,7R,8S,9R)-1,2epoxy-10-hydroxy-6-(2-methylbutyryloxy)-9-isobutyloxy-5-oxogermacran-8,12-olide. Carlipsine G (7) was isolated as colorless monoclinic crystals. The molecular formula was determined as C24H36O9 by HRESIMS and 13C NMR data, corresponding to seven

Figure 2. NOESY correlations of compounds 1 and 5.

data of 4 and 2 revealed that they possessed the same structural backbone. When compared with 2, a 2-methylbutyryloxy rather than a 3-methylbutyryloxy group was located at C6 in 4 based upon the HMBC correlations from H-6 (δH 5.11) to C-1″ (δC 176.1). Therefore, based on biosynthetic considerations,4 the structure and absolute configuration of carlipsine D (4) was assumed as (2S,4R,5R,6R,7R,8S,9R,10S)2,5-epoxy-5,10-dihydroxy-6-(2-methylbutyryloxy)-9-isobutyloxygermacran-8,12-olide. Carlipsine E (5) was obtained as colorless orthorhombic crystals, and its molecular formula was determined to be C24H32O9 by HRESIMS and 13C NMR data, requiring nine indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3484 cm−1) and carbonyl groups (1779 and 1726 cm−1). The 13C NMR data (Table 1) indicated the presence of an isobutyloxy group based upon resonances at δC 176.7, 34.1, 18.5, and 19.1. The 1H−1H COSY data (Figure 1) showed five coupled spin systems: H-2/H2-3/H-4/H3-15, H6/H-7, H-8/H-9, H3-3′/H-2′/H3-4′, and H-3″/H3-4″. HMBC correlations (Figure 1) from H-3″ (δH 6.25) to C-2″ (δC 125.6) and from H3-5″ (δH 1.95) to C-1″ (δC 166.0) and C-2″ indicated the presence of an angeloyloxy group. The HMBC data also showed correlations of H2-3 (δH 2.35)/C-1 (δC 60.2), H-1 (δH 2.70)/C-14 (δC 23.9), H-1 (δH 2.70)/C-10 (δC 70.9), H3-14 (δH 1.32)/C-9 (δC 75.4), H-6 (δH 4.86)/C-5 (δC 208.9), and H-4 (δH 3.16)/C-5, implying a 10-membered ring with the methyl groups being located at C-10 and C-4, respectively. The presence of a lactone moiety was established D



Article

Figure 4. Perspective ORTEP drawing for compounds 1 and 2. Figure 5. Perspective ORTEP drawing for compounds 5 and 7.

indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3481 cm−1) and carbonyl groups (1769 and 1743 cm−1). The 1H NMR data (Table 2) of 7 were highly similar to those of the known incuspirofide E,22 suggesting that they might share the same skeleton. Such a premise was confirmed by the 1H−1H COSY correlations (Figure 1) of H315/H-4/H2-3/H2-2/H-1, H-8/H-9, and H-6/H-7, as well as the HMBC correlations (Figure 1) from H3-14 (δH 1.07) to C1 (δC 79.9) and C-9 (δC 76.5), H3-15 (δH 1.11), H-1 (δH 4.10), and H-6 (δH 5.13) to C-5 (δC 100.2), H-7 (δH 3.25) to C-8 (δC 77.8), and H2-13 (δH 6.31 and 5.70) to C-7 (δC 44.2), C-11 (δC 133.7), and C-12 (δC 168.6). Apart from the assigned 15 carbons, the remaining signals were ascribed to a 3methylbutyryloxy and an isobutyloxy group. The NOESY correlations (Figure 3) of H-7/H-9, H-7/H-4, and H-9/Me-14 suggested that these protons and methyl groups were βoriented. The α-orientations of H-8 and H-6 were established by the cross-peak of H-6/H-8. The absolute configuration of 7 was confirmed by Cu Kα X-ray crystallographic data analysis (CCDC 1878003, Figure 5). Therefore, the structure of carlipsine G (7) was defined as (1R,4R,5S,6R,7R,8S,9R,10S)1,5-epoxy-5,10-dihydroxy-6-(3-methylbutyryloxy)-9-isobutyloxygermacran-8,12-olide. The HRESIMS and 13C NMR data of carlipsine H (8) showed a molecular formula of C25H38O9. The NMR data (Table 2) of 8 resembled those of 7, except that the isobutyloxy signals of 7 were replaced by those of a 2methylbutyryloxy group in 8. HMBC correlations from H-9

(δH 5.03) to C-1′ (δC 176.4) supported the location of the 2methylbutyryloxy group at C-9. Therefore, based on biosynthetic considerations,4 the structure and absolute configuration of carlipsine H (8) was assumed as (1R,4R,5S,6R,7R,8S,9R,10S)-1,5-epoxy-5,10-dihydroxy-9-(2methylbutyryloxy)-6-(3-methylbutyryloxy)germacran-8,12olide. Carlipsine I (9) was also isolated as colorless orthorhombic crystals and assigned a molecular formula of C24H36O9 from the HRESIMS and 13C NMR data, implying seven indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3436 cm−1) and carbonyl groups (1743 and 1723 cm−1). A 3-methylbutyryloxy and an isobutyloxy group were identified from the NMR data (Table 2). The 1H−1H COSY correlations coupled with the HSQC data revealed the presence of two spin systems of H3-15/H-4/H2-3/H2-2/H-1 and H-6/H-7/H-8/H-9 (Figure 1). HMBC correlations (Figure 1) from H3-14 (δH 1.19) to C-10 (δC 79.9), C-1 (δC 88.1), and C-9 (δC 80.8), from H3-15 (δH 1.08) to C-5 (δC 206.9), and from H-6 (δH 5.52) to C-5 (δC 206.9) and C-7 (δC51.0) established a 10-membered ring of a typical germacranolide, with Me-14 and Me-15 being located at C10 and C-4, respectively. An epoxy moiety was established by the HMBC correlations from H-1 (δH 3.92) to C-8 (δC 79.9). Two partial structural segments (C-7/C-11/C-12, C-11/C-13) were deduced from the HMBC correlations from H2-13 (δH E


Journal of Natural Products Table 2. 1H and

13

Article

C NMR Data for Compounds 6−10 in CDCl3 (δ in ppm, J in Hz)

6

7

8

9

δH, mult (J in Hz)

δH, mult (J in Hz)

δH, mult (J in Hz)

δH, mult (J in Hz)

no.

δC

1

60.3, CH

2.67, m

79.9, CH

2

53.1, CH

3.27, m

26.2, CH2

3

33.7, CH2

26.3, CH2

4 5 6 7 8 9 10 11 12 13


1.58, m 2.31, m 3.12, m

14 15 1′ 2′ 3′

23.8, CH3 19.0, CH3 176.7, C 34.1, CH 18.4, CH3

4′ 5′ 1″ 2″ 3″ 4″ 5″

4.81, 3.21, 4.57, 4.75,

d d d d

(10.5) (10.5) (8.2) (8.2)

6.42, d (1.5) 5.88, d (1.5) 1.31, s 1.21, d (7.0)

δC


4.10, dd (12.8, 5.9) 1.94, m 1.30, m 1.44, m 1.68, m 1.97, m 5.13, 3.25, 5.11, 5.00,

m m m d (8.7)

6.31, d (2.5) 5.70, d (2.5) 1.07, s 1.11, d (6.6)

2.67, m 1.20, d (6.9)

22.3, CH3 18.1, CH3 176.9, C 34.2, CH 19.2, CH3

19.1, CH3

1.28, d (7.9)

19.1, CH3

1.21, d (7.0)

171.7, C 42.8, CH2 25.5, CH 22.5, CH3 22.6, CH3

2.26, 2.08, 0.96, 0.95,

172.1, C 43.3, CH2 25.3, CH 22.5, CH3 22.5, CH3

2.26, 2.09, 0.91, 0.94,

m m d (6.6) d (6.6)

2.71, m 1.24, d (7.0)

m m d (6.5) d (6.5)

δC 80.0, CH

4.12, dd (12.8, 6.0) 1.97, m 1.31, m 1.46, m 1.69, m 1.97, m

26.2, CH2 26.3, CH2 34.2, CH 100.4, C 79.6, CH 44.2, CH 77.7, CH 76.5, CH 77.4, C 133.6, C 168.4, C 127.1, CH2

5.10, 3.24, 5.09, 5.03,

m m m d (8.2)

6.34, d (2.8) 5.74, d (2.8) 1.09, s 1.13, d (6.6)

22.6, CH3 18.1, CH3 176.4, C 41.3, CH 26.7, CH2 11.8, CH3 16.9, CH3 172.0, C 43.3, CH2 25.3, CH 22.5, CH3 22.5, CH3

6.50 and 5.93) to C-7 (δC 51.0), C-11 (δC 135.6), and C-12 (δC 169.9). The isobutyloxy and 3-methylbutyryloxy groups were assigned at C-9 (δC 80.8) and C-6 (δC 78.4), respectively, based on the HMBC correlations from H-9 (δH 5.28) to C-1′ (δC 176.0), and H-6 (δH 5.52) to C-1″ (δC 171.2). The correlations of H-7/H-9, H-9/Me-14, and H-7/H-4 revealed that they were cofacial and β-oriented, while the correlation of H-6/H-8 suggested that they were α-oriented (Figure 3). A single crystal of 9 was obtained, and the X-ray crystallographic data using Cu Kα radiation defined the absolute configuration (CCDC 1874801, Figure 6). Therefore, the structure of carlipsine I (9) was defined as (1R,4R,6R,7R,8S,9R,10S)-1,8epoxy-10-hydroxy-6-(2-methylbutyryloxy)-9-isobutyloxy-5-oxogermacrane. The HRESIMS and 13C NMR data of carlipsine J (10) suggested a molecular formula of C24H34O9. The NMR data of 10 closely resembled those of 9 except that an angeloyloxy group rather than the 3-methylpropanoyloxy group in 9 was observed in 10, which was supported by the HMBC correlations from H-3″ (δH 6.07) to C-1″ (δC 165.6), C-4″ (δC 15.8), and C-5″ (δC 20.4). An HMBC correlation from H6 (δH 5.54) to C-1″ verified the location of the angeloyloxy group at C-6. The NOESY correlations of H-3′′/Me-5′′ suggested a Z-configuration of the Δ(2″,3′′) olefinic bond in the angeloyloxy group. Therefore, based on biosynthetic considerations,4 the structure and absolute configuration of carlipsine

2.51, 1.81, 1.52, 0.95, 1.21,

m m m d (6.8) d (6.8)

2.26, 2.11, 0.98, 0.93,

m m d (7.0) d (7.0)

δC 88.1, CH 31.8, CH2 32.5, CH2 42.4, CH 206.9, C 78.4, CH 51.0, CH 79.9, CH 80.8, CH 79.9, C 135.6, C 169.9, C 131.7, CH2 19.9, CH3 21.1, CH3 176.0, C 34.0, CH 19.0, CH3

10 δC

3.92, dd (12.6, 4.2) 1.84, m 1.51, m 1.74, m

88.2, CH

2.90, m


5.52, 3.60, 4.27, 5.28,

d (10.3) m m d (6.5)

6.50, d (1.0) 5.93, d (1.0) 1.19, s 1.08, d (6.7)

31.8, CH2 32.5, CH2

δH, mult (J in Hz) 3.92, dd (12.8, 4.1) 1.86, m 1.54, m 1.74, m 2.94, m 5.54, 3.70, 4.30, 5.34,

d (10.6) m m d (6.3)

6.51, d (1.0) 5.94, d (1.0) 1.19, s 1.08, d (6.7)

2.57, m 1.14, d (7.0)

19.7, CH3 21.0, CH3 175.8, C 33.9, CH 18.9, CH3

2.56, m 1.13, d (7.1)

19.0, CH3

1.11, d (7.0)

18.8, CH3

1.11, d (7.1)

171.2, C 43.3, CH2 25.7, CH 22.4, CH3 22.5, CH3

2.15, 2.01, 0.89, 0.89,

165.6, C 126.6, C 140.2, CH 15.8, CH3 20.4, CH3

6.07, m 1.93, m 1.82, m

m m d (6.5) d (6.5)

Figure 6. Perspective ORTEP drawing for compound 9.

J (10) was assumed as (1R,4R,6R,7R,8S,9R,10S)-1,8-epoxy-10hydroxy-6-angeloyloxy-9-isobutyloxy-5-oxo-germacrane. In addition to the 10 new compounds, 12 known compounds were also isolated and identified as (2R,5S)cardivarolide A (11),4 2α,5-epoxy-5,10-dihydroxy-6α-angeloyloxy-9β-(3-methylbutyloxy)germacran-8α,12-olide (12),21 divaricin A (13),21 2β,5-epoxy-5,10-dihydroxy-6α-angeloyloxy9β-isobutyloxygermacran-8α,12-olide (14),21 ineupatolide (15),23 cernuumolide H (16),7 divaricin B (17),21 2α,5F



Article

PDA detector using a Waters Sunfire RP C18, 5 μm, 10 × 250 mm column. MCI gel CHP20P (75−150 μm, Mitsubishi Chemical Industries, Tokyo, Japan), ODS gel AAG12S50 (12 nm, S-50 μm, YMC, Japan), silica gel (100−200, 200−300, and 300−400 mesh; Qingdao Marine Chemical Industrials, Qingdao, Shandong, China), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography. TLC was carried out on precoated silica gel 60 F254 aluminum sheets (Merck, Germany). Plant Material. The whole plants of C. lipskyi were collected from Sichuan Province, People’s Republic of China, in 2016, and identified by Professor Jingui Shen from the Shanghai Institute of Materia Medica. A voucher (No. 20161114) was deposited at the Herbarium of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. Whole plants of C. lipskyi (20 kg) were extracted with 95% ethanol (3 × 60 L, 7 days each) at room temperature. The percolates were combined and evaporated under reduced pressure to afford a crude extract (2.4 kg). The extract was suspended in water and partitioned successively with petroleum ether (3 × 20 L), CH2Cl2 (3 × 20 L), and EtOAc (3 × 20 L). The CH2Cl2soluble part (380 g) was subjected to an MCI column eluted with aqueous EtOH in a gradient manner (50%, 75%, and 95%), affording three fractions (Fr. 1−Fr. 3). Fr. 2 was chromatographed over an MCI column, eluted with aqueous EtOH (50%, 55%, 60%, 65%, 70%, and 75%, 10 L each), to give nine fractions (Fr. 2A−2I). Fr. 2D was subjected to a Sephadex LH-20 column (eluted with CHCl3/MeOH 1:1) to give subfractions 2D1−2D4. Fr. 2D2 (20 g) was separated by using a silica gel column (200−300 mesh) eluted with a mixture of petroleum ether/EtOAc (3:1, 2:1, 1:1) to afford subfractions Fr. 2D2C−Fr. 2D2K. Fr. 2D2H (864 mg) was purified by a Sephadex LH-20 column (eluted with MeOH) and then preparative HPLC (CH3CN/H2O: 0−120 min, from 40% to 65%) to afford compounds 1 (10 mg), 2 (22 mg), 3 (13 mg), and 4 (18 mg). Subfractions Fr. 1A−Fr. 1H were yielded from Fr. 1 by using an ODS gel column eluted with aqueous MeOH (30%, 40%, 50%, 60%, 70%, 80%, and 100%). Fr. 1G (36 g) was separated by Sephadex LH-20 column chromatography, with CHCl3/MeOH (1:1) as mobile phase, to give three subfractions (Fr. F1G1−F1G3). Fr. 1G2 was subjected to silica gel column (300−400 mesh) chromatography using a gradient solvent system of petroleum ether/EtOAc (4:1, 3:1, 2:1, 1:1) to obtain seven subfractions (Fr. 1G2A−1G2G). Compounds 5 (26 mg) and 6 (15 mg) were isolated from Fr. 1G2E by preparative HPLC using CH3CN/H2O (0−120 min, from 45% to 70%). Fr. 1F (37 g) was separated into seven additional fractions (1F1−1F7) by a silica gel column eluted with a gradient solvent system of petroleum ether/ EtOAc (4:1, 3:1, 2:1, 1:1). Compounds 7 (21 mg) and 8 (2 mg) were obtained from Fr. 1F3 (180 mg) by preparative HPLC using CH3CN/H2O as the mobile phase (0−120 min, from 35% to 70%). Compounds 9 (40 mg) and 10 (10 mg) were obtained from Fr. 1C (8 g) by CC over a Sephadex LH-20 column (CHCl3/MeOH, 1:1) and then preparative HPLC (0−120 min, from 30% to 60%) and semipreparative HPLC (0−60 min, from 35% to 55%). Carlipsine A (1): colorless, monoclinic crystals from MeOH; mp 170−172 °C; [α]20D 0 (c 0.6, MeOH); IR (KBr) νmax 3479, 2965, 2935, 2873, 1774, 1729, 1474, 1373, 1294, 1188, 1168, 1123, 1034, 818, 731 cm−1; 1H and 13C NMR, see Table 1; ESIHRMS m/z 483.2600 [M + H]+ (calcd for C25H39O9, 483.2594). Carlipsine B (2): colorless, monoclinic crystals from MeOH; mp 214−216 °C; [α]20D 0 (c 0.4, MeOH); IR (KBr) νmax 3479, 2968, 2935, 2873, 1779, 1746, 1723, 1454, 1292, 1264, 1230, 1163, 1132, 1025, 922, 736 cm−1; 1H and 13C NMR, see Table 1; ESIHRMS m/z 469.2441 [M + H]+ (calcd for C24H37O9, 469.2438). Carlipsine C (3): white powder; [α]20D +4 (c 0.6, MeOH); IR (KBr) νmax 3481, 2965, 2937, 2879, 1777, 1732, 1465, 1373, 1292, 1266, 1236, 1188, 1126, 1039, 739 cm−1; 1H and 13C NMR, see Table 1; ESIHRMS m/z 483.2594 [M + H]+ (calcd for C25H39O9, 483.2594). Carlipsine D (4): white powder; [α]20D −5 (c 0.5, MeOH); IR (KBr) νmax 3470, 2971, 2932, 2873, 1774, 1749, 1723, 1460, 1342, 1266, 1236, 1132, 1022, 994, 933, 736 cm−1; 1H and 13C NMR, see

epoxy-5,10-dihydroxy-6α-angeloyloxy-9β-isobutyloxygermacran-8α,12-olide (18),21 cernuumolide I (19),7 cernuumolide J (20),7 (4R*,5R*,6S*,8R*,9R*)-4,8-dihydroxy-5-(2-methylpropanoyloxy)-9-(3-methylbutyryloxy)-3-oxogermacran-6,12olide (21),24 and incaspitolide D (22),24 by comparing their spectroscopic data with reported data. The cytotoxicity of all the compounds against A549 and HL60 cell lines was evaluated by using the CCK-8 assay with doxorubicin as a positive control (Table 3). Among them, Table 3. Cytotoxic Activities of Compounds 6, 7, 8, 19, 20, 21, and 22 against A549 and HL-60 Cell Lines IC50 (μM) compound

A549

6 7 8 19 20 21 22 doxorubicin

>10 10.3 ± 0.3 6.7 ± 0.4 5.2 ± 2.0 4.7 ± 0.6 6.2 ± 0.2 4.0 ± 0.8 0.4 ± 0.1

HL-60 2.8 6.2 5.6 5.0 3.0 5.5 5.9 0.2

± ± ± ± ± ± ± ±

0.3 0.2 0.5 0.4 0.5 0.9 0.5 0.0

compounds 6, 7, 8, 19, 20, 21, and 22 exhibited moderate cytotoxicity against A549 and HL-60 cells with IC50 values varying from 2.82 to 10.3 μM. The results showed that the germacranolides possessing a 10-membered ring with a carbonyl group, a 1,2-epoxy moiety, and a 1,5-hemiacetal linkage exhibited more potent cytotoxicity when compared with those containing a 2,5-hemiacetal-linked moiety. Compounds without the five-membered lactone moiety also exhibited no inhibitory activity. In summary, the systematic investigation of C. lipskyi led to the isolation of 22 germacranolides including 10 new and 12 known analogues. Based on the common 10-membered ring, these germacranolide analogues are different as far as substituents and oxygenation levels are concerned. Compounds 1−4 possess a 2,5-hemiacetal-linked moiety, presumably derived from an ordinary germacrane. Compounds 5 and 6 containing a 1,2-epoxy ring system are rare in Carpesium plants. Compounds 7 and 8 have a rare 1,5-hemiacetal linkage, which was confirmed by X-ray diffraction experiments. Similarly, the 1,8-epoxy-linked moiety of compounds 9 and 10, the first to be reported for the germacranolides, was confirmed by X-ray diffraction experiments.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a RudulphAutopol VI automatic polarimeter. IR spectra were recorded with a Nicolet Magna FTIR-750 spectrometer. NMR spectra were recorded on BrukerAvance III-500 and BrukerAvance III-600 spectrometers and a Varian MR-400 spectrometer. HRESIMS data were acquired on a Waters Synapt G2-Si Q-TOf mass detector. Single-crystal X-ray diffraction measurements were conducted on a Bruker Smart Apex II diffractometer with a graphite monochromator. LCESIMS analysis was performed on a Waters e2695 system equipped with a 2998 PDA detector, a Waters 2424 ELS, and Waters 3100 MS detectors. UPLC analysis was done on a Waters Acquity Uplc system with ELS detector, PDA detector, sample manager, and binary solvent manager. Preparative HPLC was done on a Varian PrepStar system with an Alltech 3300 ELSD using a Waters Sunfire RP C18, 5 μm, 30 × 150 mm column. Semipreparative HPLC was performed on a Waters 2690 HPLC instrument with a 996 G



Article

The final R1 was 0.0866 (I > 2σ(I)) and wR2 was 0.2857. Flack parameter: 0.05(16), Hooft parameter: 0.01(16). Crystallographic data for 5 have been deposited at the Cambridge Crystallographic Data Centre as deposit no. CCDC 1875785. Crystal Data for Compound 7. The crystal was kept at 220 K during data collection. C24H36O9 (M = 468.53 g/mol): monoclinic, space group P21 (no. 4), a = 5.5190(4) Å, b = 25.0743(18) Å, c = 9.1041(7) Å, α = 90°, β = 97.557(3)°, γ = 90°, V = 1248.93(16) Å3, Z = 2, T = 220 K, μ(Cu Kα) = 0.787 mm−1, F = 504.0, Dcalc = 1.246 g/ cm3, 19 386 reflections measured (10.418° ≤ 2σ ≤ 141.232°), 4399 unique (Rint = 0.0498, Rsigma = 0.0364), which were used in all calculations. The final R1 was 0.0714 (I > 2σ(I)) and wR2 was 0.1915. Flack parameter: 0.18(10), Hooft parameter: 0.20(7). Crystallographic data for 7 have been deposited at the Cambridge Crystallographic Data Centre as deposit no. CCDC 1878003. Crystal Data for Compound 9. The crystal was kept at 173 K during data collection. C24H38O10 (M = 486.54 g/mol): orthorhombic, space group P212121 (no. 19), a = 20.2170(10) Å, b = 5.6766(3) Å, c = 22.7143(11) Å, α = 90°, β = 90°, γ = 90°, V = 2606.8(2) Å3, Z = 4, T = 173 K, μ(Cu Kα) = 0.801 mm−1, F = 1048.0, Dcalc = 1.240 g/cm3, 44 502 reflections measured (5.852° ≤ 2σ ≤ 142.178°), 4889 unique (Rint = 0.0423, Rsigma = 0.0189), which were used in all calculations. The final R1 was 0.0396 (I > 2σ(I)) and wR2 was 0.1042. Flack parameter: −0.01(4), Hooft parameter: 0.02(4). Crystallographic data for 9 have been deposited at the Cambridge Crystallographic Data Centre as deposit no. CCDC 1874801. Cytotoxicity Assays. The Cell Counting Kit 8 (CCK-8) assay was adopted to evaluate the cytotoxicity of the compounds against A549 and HL-60 cells.25 Cells seeded in 96-well plates were incubated in humidified air containing 5% CO2 at 37 °C overnight. Appropriate dilutions of the test compounds were added to the cultures and incubated for another 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, then measured at 450 nm using a multiwell spectrophotometer (SpectraMax, Molecular Devices, USA). The cytotoxicity of compounds was expressed as an IC50, determined by the Logit method. Doxorubicin was used as a positive control.

Table 1; ESIHRMS m/z 469.2432 [M + H]+ (calcd for C24H37O9, 469.2438). Carlipsine E (5): colorless, orthorhombic crystals from MeOH; mp 193−196 °C; [α]20D +5 (c 0.7, MeOH); IR (KBr) νmax 3484, 2979, 2937, 1779, 1726, 1465, 1381, 1266, 1232, 1148, 1115, 1048, 871, 734 cm−1; 1H and 13C NMR, see Table 1; ESIHRMS m/z 465.2132 [M + H]+ (calcd for C24H33O9, 465.2125). Carlipsine F (6): white powder; [α]20D +2 (c 0.7, MeOH); IR (KBr) νmax 3493, 2977, 2937, 2873, 1779, 1746, 1723, 1465, 1370, 1264, 1191, 1154, 1118, 922, 873 cm−1; 1H and 13C NMR, see Table 2; ESIHRMS m/z 467.2286 [M + H]+ (calcd for C24H35O9, 467.2281). Carlipsine G (7): colorless, monoclinic crystals from MeOH; mp 163−164 °C; [α]20D −18 (c 0.5, MeOH); IR (KBr) νmax 3481, 2967, 2950, 2873, 1769, 1744, 1470, 1391, 1369, 1300, 1275, 1255, 1191, 1161, 1114, cm−1; 1H and 13C NMR, see Table 2; ESIHRMS m/z 469.2433 [M + H]+ (calcd for C24H37O9, 469.2438). Carlipsine H (8): white powder; [α]20D −5 (c 0.5, MeOH); IR (KBr) νmax 3456, 2963, 2932, 2876, 1771, 1737, 1463, 1373, 1278, 1179, 1151, 1115, 1090, 1036 cm−1; 1H and 13C NMR, see Table 2; ESIHRMS m/z 483.2603 [M + H]+ (calcd for C25H39O9, 483.2594). Carlipsine I (9): colorless, orthorhombic crystals from MeOH; mp 185−187 °C; [α]20D +33 (c 0.5, MeOH); IR (KBr) νmax 3436, 2965, 2931, 2879, 1743, 1723, 1622, 1465, 1389, 1294, 1165, 1028 cm−1; 1 H and 13C NMR, see Table 2; ESIHRMS m/z 469.2424 [M + H]+ (calcd for C24H37O9, 469.2438). Carlipsine J (10): white powder; [α]20D +110 (c 0.3, MeOH); IR (KBr) νmax 3456, 2963, 2932, 2876, 1771, 1737, 1463, 1373, 1278, 1179, 1151, 1115, 1090, 1036 cm−1; 1H and 13C NMR, see Table 2; ESIHRMS m/z 467.2258 [M + H]+ (calcd for C24H35O9, 467.2281). X-ray Crystallographic Analysis of Compounds 1, 2, 5, 7, and 9. Crystals were obtained from their MeOH solutions, respectively, and suitable crystals were selected for the X-ray crystallographic analysis. Using Olex2, the structures were solved with the ShelXT structure solution program using Intrinsic Phasing and refined with the ShelXL refinement package using Least Squares minimization. Copies of crystallographic data of every crystal can be obtained free of charge via the Internet at www.ccdc.cam.ac.uk/conts/ retrieving.html or on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [tel: (+44) 1223-336-408; fax: (+44) 1223-336-033; e-mail: [email protected]]. Crystal Data for Compound 1. The crystal was kept at 220 K during data collection. C24H36O9: M = 468.53 g/mol, monoclinic, space group P21 (no. 4), a = 5.5190(4) Å, b = 25.0743(18) Å, c = 9.1041(7) Å, α = 90°, β = 97.557 (3)°, γ = 90°, V = 1248.93(16) Å3, Z = 2, T = 220 K, μ(Cu Kα) = 0.787 mm−1, F = 540.0, Dcalc = 1.246 g/cm3, 19 386 reflections measured (10.418° ≤ 2σ ≤ 141.232°), 4399 unique (Rint = 0.0498, Rsigma = 0.0364), which were used in all calculations. The final R1 was 0.0714 (I > 2σ(I)) and wR2 was 0.1915. Flack parameter: 0.07(5), Hooft parameter: 0.06(6). Crystallographic data for 1 have been deposited at the Cambridge Crystallographic Data Centre as deposit no. CCDC 1875786. Crystal Data for Compound 2. The crystal was kept at 173 K during data collection. C24H38O10 (M = 486.54 g/mol): monoclinic, space group P21 (no. 4), a = 9.1278(12) Å, b = 10.442(4) Å, c = 14.160(4) Å, α = 90°, β = 105.475(12)°, γ = 90°, V = 1300.7(6) Å3, Z = 2, T = 173.0 K, μ(Cu Kα) = 0.803 mm−1, F = 524.0, Dcalc = 1.242 g/cm3, 13 367 reflections measured (10.41° ≤ 2σ ≤ 136.676°), 4709 unique (Rint = 0.0315, Rsigma = 0.0323), which were used in all calculations. The final R1 was 0.0416 (I > 2σ(I)) and wR2 was 0.1146. Flack parameter: 0.18(7), Hooft parameter: 0.18(7). Crystallographic data for 2 have been deposited at the Cambridge Crystallographic Data Centre as deposit no. CCDC 1875787. Crystal Data for Compound 5. The crystal was kept at 296 K during data collection. C24H32O9 (M = 464.49 g/mol): orthorhombic, space group P212121 (no. 19), a = 5.9654(2) Å, b = 17.2094(6) Å, c = 24.7971(9) Å, α = 90°, β = 90°, γ = 90°, V = 2545.69(15) Å3, Z = 4, T = 296 K, μ(Cu Kα) = 0.772 mm−1, F = 992.0, Dcalc = 1.212 g/cm3, 15 317 reflections measured (6.252° ≤ 2σ ≤ 130.412°), 4168 unique (Rint = 0.0547, Rsigma = 0.0493), which were used in all calculations.

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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.8b01004. 1D and 2D NMR and IR spectra, HRESIMS data of new compounds (1−10), structures of known compounds (11−22), and X-ray data for compounds 1, 2, 5, 7, and 9 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H. Liu). *E-mail: [email protected] (Y. Ye). 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 of China “Key New Drug Creation and Manufacturing Program” (2015ZX09103002). Our thanks are also given to the National Natural Science Foundation of China (81573305, 81673327) and the International Partnership Program of Chinese Academy of Sciences H



Article

(153631KYSB20160004, 153631KYSB20170043). Financial support from the Collaborative Innovation Center of Southwest Ethnic Medicine, Guangxi Normal University (CICSEM 2017-A), is also acknowledged.

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REFERENCES

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