Bisabolane-Type Sesquiterpenoids from the Whole Plant of

DOI: 10.1021/acs.jnatprod.5b00380. Publication Date (Web): July 22, 2015 ... Society and American Society of Pharmacognosy. *Phone: 86 27 83657870...
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Bisabolane-Type Sesquiterpenoids from the Whole Plant of Parasenecio rubescens An Jin,† Wen-Ming Wu,† Heng-Yi Yu,‡ Ming Zhou,† Ye Liu,† Tian Tian,† and Han-Li Ruan*,† †

Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Hangkonglu 13, Wuhan 430000, People’s Republic of China ‡ Department of Pharmacy, Tongji Hospital Affiliated Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China S Supporting Information *

ABSTRACT: Twenty-two new highly oxygenated bisabolanetype sesquiterpenoids, pararubin A−V (1−22), were isolated from the whole plant of Parasenecio rubescens. The structure determination of 1−22, including the assignment of their relative configurations, was accomplished by extensive 1D and 2D NMR spectroscopy. The absolute configuration of pararubin A (1) was established by single-crystal X-ray crystallographic analysis. The isolated compounds were evaluated for their cytotoxic effects against three cancer cell lines (B16 mouse melanoma cells, HepG2 human hepatocellular carcinoma cells, and MCF7 human breast adenocarcinoma cells) and for their antimicrobial effects against Staphylococcus aureus, Escherichia coli, and Monilia albicans.

T

he plant genus Parasenecio (Compositae) comprises approximately 80 species, 51 of which are native to China.1 Some species of the genus have been used as folk medicines in China for their anticough, antibacterial, and anti-inflammatory activities.2 Previous studies on the genus Parasenecio led to the isolation of a variety of sesquiterpenoids, including guaianes, eremophilanes, and germacranes, some of which showed promising biological properties such as cytotoxic and antimicrobial activities.3−9 Parasenecio rubescens is a perennial herb, which is mainly distributed in the Jiangxi, Hunan, Anhui, and Fujian Provinces in China. This herb has been used for the treatment of tussis, tonsillitis, and tuberculosis in Chinese folk medicines.10 The chemical components of P. rubescens have not been reported hitherto. In our continuing search for bioactive terpenoids from Chinese herbs, the acetone extract of the whole plant of P. rubescens was investigated. As a result, 22 new highly oxygenated bisabolane-type sesquiterpenoids, named pararubins A−V (1−22), were isolated. This represents the first isolation of bisabolane-type sesquiterpenoids from the genus Parasenecio. Herein, the isolation and structural elucidation of the new compounds are described as well as their cytotoxic effects against three cancer cell lines (B16 mouse melanoma cells, HepG2 human hepatocellular carcinoma cells, and human breast adenocarcinoma cells) and their antimicrobial effects against Staphylococcus aureus, Escherichia coli, and Monilia albicans.

■

ether and EtOAc. The EtOAc-soluble fraction was repeatedly subjected to a series of chromatographic methods to afford 22 new bisabolane-type sesquiterpenoids (1−22) (Figure 1). The six-membered rings of 1−13, 14 and 15, and 16−22 possess the same relative configurations, respectively. Pararubin A (1) was obtained as white needles, and its molecular formula was determined as C27H42O11 by the HRESIMS analysis and 13C NMR data, requiring seven indices of hydrogen deficiency. The 1H (Table 1) and 13C (Table 2) NMR data of 1 showed the presence of two angeloyloxy groups, a terminal double bond, a methylene group, and seven methine groups including six oxygenated ones. The 1H−1H COSY correlations (Figure 2) of 1 exhibited two main structural sequences, −CH(H-2)−CH(H-1)−CH(H-6)−CH(H-5)− CH(H-4)− and −CH(H-8)−CH2(H-9)−CH(H-10)−, which were connected to form a bisabolane sesquiterpenoid skeleton by the following HMBC correlations (Figure 2): H-1, H-5/C-7; H-2, H-4/C-15; H-6, H-8/C-14; H-10/C-12; and H-12/C-13. The attachment of an acetoxy and two angeloyl groups could also be determined by the HMBC correlations of H-2/C-1′, H-4/C-1″, and H-10/C-1‴, respectively. The relative configuration of the six-membered ring of 1 was established by the 1H NMR coupling constants: H-6 is assumed to be β-oriented, then H-1 and H-5 should be α-axial, based on the large coupling constants of H-1/H-6 (J1, 6 = 11.4 Hz) and H-5/H-6 (J5, 6 = 11.4 Hz). H-2 and H-4 must be α-equatorial due to the small coupling constants (J1, 2, J4, 5 = 3.5 Hz).

RESULTS AND DISCUSSION

The acetone extract of the whole plant of P. rubescens was dissolved in water and sequentially partitioned with petroleum © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 30, 2015

A

DOI: 10.1021/acs.jnatprod.5b00380 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of compounds 1−22.

Pararubin I (9), a colorless gum, has a molecular formula of C28H44O11 with seven indices of hydrogen deficiency according to the HRESIMS and 13C NMR data. The 1D (Tables 2 and 3) and 2D (Supporting Information Figures S67−S70) NMR data of 9 were similar to those of 3. The only difference between the two compounds was the presence of a methoxy group at C-11 in 9, which was confirmed by the HMBC correlation of MeO-11/C-11. Using the same methods as those applied for 1 permitted the assignment of the relative configuration of 9. Therefore, the structure of 9 (pararubin I) was defined as shown. Pararubins J−L (10−12) were all obtained as colorless gums, and their molecular formulas were determined as C25H40O10 with six indices of hydrogen deficiency by their respective HRESIMS and 13C NMR data. Comparing their NMR spectroscopic data (Tables 2 and 3) with those of 1 revealed that 10−12 were bisabolane-type sesquiterpenoids with only two angeloyloxy groups each. The two angeloyloxy groups of pararubin J (10) were located at C-4 and C-10 via the HMBC correlations of H-4/C-1′ and H-10/C-2″. The relative configuration of 10 was established using the same methods as those applied for 1. Consequently, the structure of 10 was elucidated as shown. Similarly, the structures of pararubins K (11) and L (12) were respectively confirmed as shown by comparing their 1D (Tables 2 and 3) and 2D (Supporting Information Figures S81−S96) NMR data with those of 10. Pararubin M (13) has the molecular formula of C31H46O10 with nine indices of hydrogen deficiency by HRESIMS and 13C NMR data. Comparison of the NMR data (Tables 2 and 3) of 13 with those of 1 revealed a similar bisabolane-type sesquiterpenoid skeleton, with the major difference being the presence of a Δ11,12 double bond in 13, which was confirmed by the HMBC correlations (Supporting Information Figure S101) of H-13/C-12 and H-13/C-10. The 1H and 13C NMR spectra

The NOESY correlations (Figure 2) were in accordance with the above observations. The absolute configuration of 1 was established as 1S, 2S, 3R, 4R, 5R, 6S, 8S, 10R, 2′Z, and 2‴Z by single-crystal X-ray diffraction using Cu Kα radiation (Figure 3). Thus, the chemical structure of 1 was established as shown and given the name pararubin A. Pararubin B (2), a colorless gum, has the same molecular formula as 1 according to the HREIMS and 13C NMR data. The NMR data (Tables 1 and 2) revealed that 2 was also a bisabolanetype sesquiterpenoid with an acetoxy and two angeloyloxy groups, the positions of which were established by the HMBC correlations of H-2/C-1′, H-4/C-1″, and H-8/C-1‴, respectively. The relative configuration of the six-membered ring of 2 was established by similar methods as those used for 1. When H-6 is assumed to be β-oriented, the large coupling constants of H-1/H-6 (J1, 6 = 11.4 Hz) and H-5/H-6 (J5, 6 = 11.2 Hz) indicated the α-axial orientations of H-1 and H-5. H-2 and H-4 must be α-equatorial because of the small coupling constants (J1, 2, J4, 5 = 3.6 Hz) observed. Thus, the structure of 2 was defined as shown. Pararubins C−H (3−8) were all isolated as colorless gums, sharing the same molecular formulas as 1 and 2, C27H42O11, based on their respective HRESIMS and 13C NMR data. Their NMR data (Tables 1 and 2) were highly similar to those of 1 and 2, revealing that 3−8 were also bisabolane-type sesquiterpenoids with an acetoxy and two angeloyloxy groups each. The positions of these groups in pararubins C−H were determined by the respective HMBC correlations of H-5/C-1″, H-1/C-1′, and H-8/C-1‴ for 3; H-5/C-1″, H-1/C-1′, and H-8/C-1‴ for 4; H-5/C-1″, H-2/C-1′, and H-10/C-1‴ for 5; H-5/C-1″, H-1/C-1′, and H-10/C-1‴ for 6; H-1/C-1′, H-4/C-1″, and H-10/C-1‴ for 7; H-1/C-1′, H-4/C-1″, and H-8/C-1‴ for 8. The relative configurations of pararubins C−H (3-8) resembled those of 1 and were defined by comparing their 1H NMR coupling constants and NOESY correlations. Thus, the structures of 3−8 were established as shown. B

DOI: 10.1021/acs.jnatprod.5b00380 J. Nat. Prod. XXXX, XXX, XXX−XXX

1 2 4 5 6 8 9a 9b 10 12 13 14a 14b 15 2′ 3′ 4′ 5′ 2″ 3″ 4″ 5″ 3‴ 4‴ 5‴

position

qq (7.2, dq (7.2, q (1.1) qq (7.2, dq (7.2, q (1.1)

6.14 2.05 2.02 6.14 1.99 1.93

C

1.1) 1.1)

1.1) 1.1)

dd (11.4, 3.5) d (3.5) d (3.5) dd (11.4, 3.5) t (11.4) dd (9.4, 4.6) m m dd (10.0, 1.5) s s s s s s

3.99 5.06 5.18 4.25 2.92 4.08 2.14 1.79 4.99 1.14 1.14 5.28 5.26 1.15 2.07

1

6.17 2.06 2.01 6.17 2.00 1.93

4.24 5.11 5.18 4.10 2.83 5.34 1.93 1.93 3.46 1.15 1.16 5.32 5.19 1.15 2.08

qq (7.2, dq (7.2, q (1.3) qq (7.2, dq (7.2, q (1.3) 1.3) 1.3)

1.3) 1.3)

dd (11.2, 3.6) d (3.6) d (3.6) dd (11.2, 3.6) t (11.2) overlap overlap overlap m s s s s s s

2

qq (7.3, 1.4) dq (7.3, 1.4) q (1.4) s

6.12 2.05 1.95 1.97

6.13 qq (7.2, 1.4) 1.98 dq (7.2, 1.4) 1.95 q (1.4)

dd (11.5, 3.5) d (3.5) d (3.5) dd (11.5, 3.5) t (11.5) overlap overlap overlap m s s s s s

4.12 5.21 3.69 5.36 3.09 5.31 1.88 1.88 3.40 1.15 1.16 5.25 5.16 1.29

3

4

qq (7.2, 1.4) dq (7.2, 1.4) q (1.4) s

6.13 qq (7.2, 1.4) 1.98 dq (7.2, 1.4) 1.95 q (1.4)

6.02 1.96 1.92 1.99

5.48 dd (11.7, 3.0) 3.71 d (3.0) 3.75 d (3.0) 5.42 dd (11.7, 3.0) 3.28 t (11.7) 5.64 d (11.0) 1.87m 1.68 m 3.33 m 1.13 s 1.15 s 5.29 s 5.20 s 1.48 s

Table 1. 1H NMR (400 MHz) Data for Compounds 1-8 (Methanol-d4, J in Hz)

6.12 1.98 1.92 6.12 2.03 1.96

qq (7.0, dq (7.0, q (1.5) qq (7.0, dq (7.0, q (1.5) 1.5) 1.5)

1.5) 1.5)

5.39 dd (11.4, 2.9) 3.67 d (2.9) 5.16 d (2.9) 4.07 dd (11.4, 2.9) 2.98 t (11.4) 4.07 m 2.37 m 1.64 m 5.03 dd (8.9, 2.5) 1.2 s 1.2 s 5.33 s 5.14 s 1.28 s 2.05 s

5

6.10 1.97 1.89 6.10 1.99 1.94

5.48 3.74 3.76 5.31 3.01 4.27 2.38 1.53 5.09 1.23 1.24 5.34 5.11 1.47 2.02

qq (7.1, dq (7.1, q (1.5) qq (7.1, dq (7.1, q (1.5)

1.5) 1.5)

1.5) 1.5)

dd (11.4, 2.9) d (2.9) d (2.9) dd (11.4, 2.9) t (11.4) m m m overlap s s s s s s

6

qq (7.1, 1.4) dq (7.1, 1.4) q (1.4) s

6.11 qq (7.1, 1.4) 2.03 dq (7.1, 1.4) 1.96 q (1.4)

6.11 1.98 1.92 2.04

5.39 dd (11.6, 3.2) 3.67 d (3.2) 5.16 d (3.2) 4.08 dd (11.6, 3.2) 2.99 t (11.6) 4.08 m 2.36 m 1.65 m 5.03 dd (8.7, 2.8) 1.2 s 1.2 s 5.33 s 5.14 s 1.29 s

7

qq (7.3, 1.5) dq (7.3, 1.5) q (1.5) s

dd (11.6, 3.1) d (3.1) d (3.1) dd (11.6, 3.1) t (11.6) dd (7.1, 5.6) m m dd (9.9, 2.1) s s s s s

6.14 qq (7.3, 1.5) 2.01 dq (7.3, 1.5) 1.96 q (1.5)

6.14 2.04 1.96 1.93

5.30 3.65 5.18 4.13 3.21 5.54 2.29 1.56 3.50 1.12 1.13 5.33 5.22 1.28

8

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00380 J. Nat. Prod. XXXX, XXX, XXX−XXX

D

169.9 129.2 140.1 16.1 20.8

169.3 129.4 138.8 16.0 20.9

169.3 129.2 140.4 16.2 21.1

73.2 77.0 73.5 74.8 75.1 41.4 149.7 77.1 37.2 75.6 73.9 25.2 25.6 111.7 24.0 169.2 129.6 139.0 16.2 20.9 172.2 21.0

3a

168.1 129.5 139.4 16.1 21.1

75.9 76.6 72.6 76.4 75.9 37.4 148.9 73.4 37.8 75.5 73.4 25.2 25.5 112.9 24.4 168.8 129.6 138.4 16.1 21.0 172.1 21.0

4a

169.3 129.6 139.1 16.1 21.1

169.3 129.6 138.6 16.0 20.9

75.3 74.9 74.0 77.4 72.7 41.6 151.7 74.6 36.5 78.8 72.9 25.3 26.8 111.1 24.0 172.3 21.1

5a

169.2 129.2 138.6 16.1 21.0

168.4 129.6 139.8 16.1 20.9

75.1 76.1 72.6 75.7 77.0 38.1 152.6 74.3 36.9 78.9 72.9 25.7 25.7 109.9 24.4 171.9 21.1

6a

169.3 129.6 139.0 16.1 21.1

75.3 75.0 73.9 77.4 72.7 41.6 151.9 74.5 36.6 78.8 72.9 25.4 26.8 111.2 24.0 169.3 129.6 138.6 16.1 20.9 172.3 21.1

7a

168.7 129.8 139.8 16.2 21.0

75.6 74.9 73.8 77.8 72.2 40.4 147.7 77.4 36.0 76.0 73.9 25.3 25.9 104.4 24.0 169.2 129.5 139.2 16.2 21.0 172.5 21.0

8a

49.3

168.0 127.4 140.3 16.0 20.6

72.3 75.8 72.4 74.8 73.0 41.4 147.7 76.2 35.8 73.5 77.0 19.4 20.8 110.9 24.0 166.6 127.3 139.6 16.1 20.8 170.6 21.0

9b 74.0 77.3 73.8 77.6 70.4 41.6 150.1 74.9 36.3 77.7 72.8 26.9 25.3 114.7 24.3 169.2 129.4 139.3 16.2 21.1 169.3 129.5 138.9 16.1 20.9

10a

Data were measured in methanol-d4 at 100 MHz. bData were measured in CDCl3 at 100 MHz.

168.9 129.4 139.5 16.2 21.2

168.7 129.3 139.7 16.2 21.2

a

69.6 77.6 72.9 76.7 71.6 46.9 149.4 76.1 37.4 75.5 73.6 25.3 25.5 113.2 23.5 172.6 21.3

72.3 77.3 72.8 77.2 70.1 41.8 149.5 74.7 36.3 77.7 73.1 25.4 26.8 114.6 23.6 172.5 21.1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 1‴′ 2‴′ 3‴′ 4‴′ 5‴′ OMe-10 OMe-11

2a

1a

position

Table 2. 13C NMR (100 MHz) Data for Compounds 1−22 71.7 78.9 72.5 76.2 75.1 43.1 149.2 74.3 38.1 75.7 73.6 25.3 25.3 114.5 24.6 168.9 129.4 139.1 16.1 21.0 169.1 129.3 139.4 16.1 20.9

11a 71.2 77.4 73.6 77.0 71.9 46.4 150.2 76.7 36.3 75.8 73.6 25.4 25.4 112.6 24.2 169.2 129.7 139.0 16.2 21.1 169.9 129.1 140.2 16.2 20.8

12a 73.2 76.8 73.9 75.1 73.5 42.2 149.7 76.8 41.1 72.8 149.3 110.7 18.5 111.1 24.1 169.3 129.7 138.7 16.2 21.1 167.3 115.4 163.4 34.6 12.3 18.9 169.3 129.1 140.5 16.2 20.8

13a 74.1 77.0 78.0 210.5 73.4 53.1 150.0 75.9 37.2 75.6 73.5 25.3 25.4 113.1 20.0 168.0 128.9 140.0 16.2 20.8 169.7 129.1 140.2 16.2 20.8

14a 74.1 77.0 78.0 210.5 74.1 49.2 149.2 74.0 37.8 75.6 74.1 25.2 25.3 114.4 20.3 168.7 129.4 139.3 16.1 20.9 168.7 129.4 139.2 16.2 20.9

15a

168.2 128.9 140.6 16.3 21.0

168.3 128.9 140.6 16.2 21.0 49.7

171.7 20.7

69.2 64.1 59.3 72.4 69.4 42.9 145.4 75.7 37.8 75.3 73.4 25.0 25.6 116.2 20.3 168.3 128.4 140.0 16.1 20.9 167.8 128.5 140.2 16.1 20.6

17a

171.1 20.7

69.4 64.1 59.3 72.4 69.5 42.7 145.4 75.8 36.9 73.6 78.2 20.2 21.6 116.2 19.1 168.2 128.5 140.1 16.1 20.9 167.8 128.5 140.0 16.0 20.6

16a

167.3 128.5 140.3 16.2 21.0 49.7

171.3 20.7

69.3 64.1 59.3 72.4 69.5 42.8 145.1 75.7 37.7 75.7 73.7 27.9 30.0 116.6 19.1 167.6 128.7 140.7 16.3 21.0 167.1 128.4 140.2 16.0 20.6

18a

167.0 127.4 139.7 16.0 20.6

171.2 20.9

67.1 63.1 57.6 71.6 67.7 44.1 142.6 74.9 35.2 75.4 72.7 24.1 26.0 116.8 18.9 167.1 127.3 139.5 16.0 20.6 170.3 20.7

19b

169.0 129.4 138.8 16.1 20.9

172.3 20.8

69.4 64.1 59.0 72.9 70.3 42.2 149.0 74.8 37.5 78.4 72.7 25.4 27.0 113.6 19.0 168.4 128.7 140.1 16.1 20.9 171.7 20.5

20a

169.2 129.5 138.8 16.0 20.9

172.6 20.9

67.7 66.9 59.1 73.2 71.2 40.4 148.9 75.2 36.3 77.9 73.0 25.8 26.5 114.8 19.2 171.8 20.6

21a

172.2 20.7

68.7 64.2 59.1 73.0 70.4 41.7 146.6 81.0 40.5 78.5 84.5 25.7 23.0 114.0 19.0 168.4 128.5 140.3 16.1 20.8 171.8 20.5

22a

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00380 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Pararubin P (16) was obtained as a yellow oil, and its molecular formula was deduced to be C33H48O11 by the HRESIMS and 13C NMR data, corresponding to ten indices of hydrogen deficiency. The 1D (Tables 2 and 4) and 2D NMR spectra (Supporting Information Figures S123−S126) of 16 exhibited signals of a bisabolane-type sesquiterpenoid skeleton with three angeloyloxy, an acetoxy, and a methoxy group, the C-1, C-4, C-8, C-5, and C-11 locations of which were confirmed by HMBC analyses, respectively. Apart from these characteristic signals, resonances for an epoxy group were also observed in the 1D NMR spectra; its position was assigned to C-2 and C-3 from the HMBC correlations of H-1/C-3 and H-6/C-2 (Figure 4). The relative configuration of the cyclohexane ring of 16 was established from the 1H NMR coupling constants and the NOESY correlations (Figure 4). When H-6 is assumed to be β-oriented, H-1 should be α-axial due to the large coupling constant of H-1/H-6 (J1, 6 = 11.3 Hz), and H-5 should be β-equatorial due to the small coupling constant (J5, 6 = 1.8 Hz). The orientation of H-4 was determined to be β-axial because of the NOESY correlation between H-4 and H-6. The coupling constant of H-1/H-2 was nearly zero and indicated that their dihedral angle was about 90° due to the 2α,3α-epoxy group (the cyclohexane ring should possess a twist-boat conformation).11 Thus, the structure of 16 was determined as shown. Pararubin Q (17), a yellow oil, exhibited a molecular formula of C32H46O11 as determined by the HRESIMS and 13C NMR data, requiring ten indices of hydrogen deficiency. Analysis of its 1D (Tables 2 and 4) and 2D NMR data (Supporting Information Figures S131−S134) revealed that the structure of 17 resembled that of 16, except for the absence of the methoxy group at C-11. Thus, the structure of 17 was elucidated as shown. Pararubin R (18) was isolated as a yellow oil. Its molecular formula was established as C33H48O11, with ten indices of hydrogen deficiency by the HRESIMS and 13C NMR data. The 1D (Tables 2 and 4) and 2D NMR data (Supporting Information Figures S139−S142) of 18 showed similarity to those of 16, except for the C-10 methoxy group in 18 based upon the

Figure 2. Key HMBC (→), 1H−1H COSY (thick lines) and NOESY (↔) correlations of compound 1.

showed the characteristic signals of a (4-methylsenecioyl)oxy and two angeloyloxy groups, the positions of which were established from the HMBC correlations of H-5/C-1″, H-2/ C-1′, and H-8/C-1‴, respectively. Analysis of the 1H NMR coupling constants and NOESY data (Supporting Information Figure S102) of 13 revealed that it has the same relative configuration as that of 1. Thus, the structure of 13 was established as shown and named pararubin M. The molecular formula of pararubin N (14) was determined to be C25H38O10 with seven indices of hydrogen deficiency on the basis of its HRESIMS and 13C NMR data. The 1D NMR data (Tables 2 and 3) of 14 were similar to those of 11, except for the presence of a carbonyl group in 14. The C-4 location of the carbonyl group was deduced from the HMBC correlations (Supporting Information Figure S109) of H-2, H-15/C-4. Moreover, the attachments of two angeloyloxy groups were also determined by the HMBC correlations. The relative configuration of 14 was identical to that of 1 on the basis of its 1H NMR coupling constants and NOESY correlations (Supporting Information Figure S110). Thus, the structure of 14 was elucidated as shown. Pararubin O (15) has the same molecular formula of C25H38O10 as 14 based on the HRESIMS and 13C NMR data. Its NMR data (Tables 2 and 3) were similar to those of 14, with the differences being the C-1 and C-8 locations of the angeloyloxy groups, as confirmed by the HMBC correlations (Supporting Information Figure S117). Therefore, the structure of 15 was proposed as shown.

Figure 3. ORTEP drawing of compound 1. E

DOI: 10.1021/acs.jnatprod.5b00380 J. Nat. Prod. XXXX, XXX, XXX−XXX

a

F

dd (11.3, 3.5) d (3.5) d (3.5) dd (11.3, 3.5) t (11.3) dd (9.3, 4.7) m m dd (10.0, 1.8) s s s s s qq (7.2, 1.5) dq (7.2, 1.5) q (1.5)

6.12 qq (7.2, 1.5) 1.99 dq (7.2, 1.5) 1.93 q (1.5)

3.89 3.54 5.18 4.21 2.92 4.09 2.19 1.79 5.00 1.13 1.14 5.29 5.27 1.30 6.12 2.03 1.96

10a

Data were measured in methanol-d4. bData were measured in CDCl3.

qq (6.9, 1.3) dq (6.9, 1.1) q (1.3) s

4.18 dd (11.5, 3.2) 5.36 d (3.2) 3.76 d (3.2) 5.42 dd (11.5, 3.2) 2.76 t (11.5) 5.12 dd (10.0, 1.4) 1.92 m 1.60 m 3.56 d (10.2) 1.08 s 1.13 s 5.21s 5.20 s 1.40 s 6.12 qq (6.9, 1.3) 2.03 dq (6.9, 1.3) 1.94 q (1.3) 2.00 s

1 2 4 5 6 8 9a 9b 10 12 13 14a 14b 15 3′ 4′ 5′ 2″ 3″ 4″ 5″ 6″ 3‴ 4‴ 5‴ OMe-11

6.12 1.97 1.89 3.20

9b

position dd (11.5, 3.0) d (3.0) d (3.0) dd (11.5, 3.0) t (11.5) dd (10.7, 1.3) m m dd (10.7, 1.5) s s s s s qq (7.3, 1.4) dq (7.3, 1.4) q (1.4)

11a

6.13 qq (7.3, 1.4) 1.98 dq (7.3, 1.4) 1.92 q (1.4)

4.22 3.65 3.74 5.36 3.01 5.57 1.88 1.75 3.41 1.11 1.14 5.34 5.21 1.49 6.06 1.96 1.88

Table 3. 1H NMR (400 MHz) Data for Compounds 9−15 (J in Hz)

6.20 qq (7.2, 1.5) 2.00 dq (7.2, 1.5) 1.93 q (1.5)

4.10 dd (11.2, 3.4) 3.57 d (3.4) 5.19 d (3.4) 4.08 dd (11.2, 3.4) 2.80 t (11.2) 5.27 m 1.99 m 1.89 m 3.45 dd (10.8, 1.6) 1.16 s 1.16 s 5.30s 5.20 s 1.31 s 6.11 qq (7.2, 1.5) 2.04 dq (7.2, 1.5) 1.96 q (1.5)

12a 4.13 dd (11.4, 3.4) 5.23 d (3.4) 3.68 d (3.4) 5.44 dd (11.4, 3.4) 3.02 t (11.4) 5.23 overlap 2.12 m 1.69 m 4.09 m 4.80 s, 4.96 s 1.74 s 5.20s 5.17 s 1.30 s 6.11 qq (7.2, 1.5) 2.04 dq (7.2, 1.5) 1.97 q (1.5) 5.62 q (1.2) 6.20 qq (7.3, 1.5) 2.15 q (7.4) 1.05 t (7.4) 2.11 overlap 6.21 qq (7.3, 1.4) 2.00 dq (7.3, 1.4) 1.95 q (1.4)

13a

6.11 qq (7.1, 1.4) 2.00 dq (7.3, 1.5) 1.93 q (1.5)

6.11 qq (7.1, 1.4) 1.97 dq (7.1, 1.4) 1.92 q (1.4)

dd (11.6, 2.7) t (11.6) dd (10.8, 1.6) m m dd (10.8, 1.6) s s s s s qq (7.1, 1.4) dq (7.1, 1.4) q (1.4)

4.98 3.00 5.61 1.87 1.78 3.38 1.11 1.14 5.37 5.27 1.35 6.08 1.97 1.90

4.96 2.62 5.33 1.89 1.89 3.43 1.15 1.16 5.38 5.30 1.22 6.11 2.04 1.86

dd (11.1, 3.0) t (11.1) dd (10.3, 2.1) overlap overlap dd (10.3, 2.1) s s s s s qq (7.2, 1.5) dq (7.2, 1.5) q (1.5)

5.80 dd (11.6, 2.7) 3.94 d (2.7)

15a

4.58 dd (11.1, 3.0) 3.94 d (3.0)

14a

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00380 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 4. 1H NMR (400 MHz) Data for Compounds 16−22 (J in Hz) 16a

position 1 2 4 5 6 8 9a 9b 10 12 13 14a 14b 15 2′ 3′ 4′ 5′ 2″ 3″ 4″ 5″ 2‴ 3‴ 4‴ 5‴ 3‴′ 4‴′ 5‴′ OMe-10 OMe-11 a

5.42 3.17 5.53 5.26 2.99 5.65 2.14 1.42 3.45 1.11 1.15 5.30 5.11 1.34

d (11.3) s d (4.6) dd (4.6, 1.8) dd (11.3, 1.8) dd (11.7, 1.6) m m dd (11.2, 1.4) s s s s s

17a

18a

5.42 d (11.3) 3.17 s 5.53 d (4.6) 5.30 dd (4.6, 1.8) 3.01 dd (11.3, 1.8) 5.65 dd (11.7, 1.6) 2.14 m 1.43 m 3.35 dd (11.2, 1.4) 1.13 s 1.15 s 5.31s 5.11 s 1.34 s

5.42 3.18 5.53 5.26 3.00 5.66 2.34 1.54 3.53 1.51 1.56 5.33 5.14 1.34

6.14 qq (7.2, 1.4) 1.98 dq (7.2, 1.4) 1.92 q (1.4)

6.15 qq (7.2, 1.4) 1.98 dq (7.2, 1.4) 1.93 q (1.4)

6.17 qq (7.2, 1.4) 2.00 dq (7.2, 1.4) 1.93 q (1.4)

6.14 1.98 1.85 2.01

6.11 1.97 1.92 2.01

6.17 1.97 1.86 2.02

qq (7.2, 1.4) dq (7.2, 1.4) q (1.4) s

qq (7.2, 1.4) dq (7.2, 1.4) q (1.4) s

d (11.3) s d (4.6) dd (4.6, 1.8) dd (11.3, 1.8) dd (11.7, 1.6) m m dd (11.2, 1.4) s s s s s

qq (7.2, 1.4) dq (7.2, 1.4) q (1.4) s

19b

20a

5.51 3.08 5.36 5.40 2.87 5.49 1.85 1.85 3.23 1.12 1.14 5.36 5.20 1.35

d (11.2) s d (4.6) dd (4.6, 1.6) dd (11.2, 1.6) overlap overlap overlap dd (9.1, 3.2) s s s s s

5.40 3.13 5.44 5.42 2.86 4.22 1.90 1.90 4.95 1.17 1.19 5.12 4.98 1.33

d (11.3) s d (4.6) dd (4.6, 1.8) dd (11.3, 1.8) dd (8.4, 5.0) overlap overlap overlap s s s s s

6.09 1.96 1.87 2.06

qq (7.3, 1.3) dq (7.3, 1.3) q (1.3) s

6.12 1.97 1.90 2.03

qq (7.2, 1.3) dq (7.2, 1.3) q (1.3) s

2.06 s

21a 4.15 3.16 5.35 5.26 2.44 4.09 2.11 1.81 4.93 1.17 1.17 5.09 5.08 1.32 2.01

22a

d (10.9) s d (4.6) dd (4.6, 1.4) dd (10.9, 1.4) dd (8.8, 4.8) m m, dd (10.0, 1.9) s s s s s s

2.03 s

2.06 s

5.41 3.14 5.38 5.46 3.08 4.41 2.41 1.81 3.98 1.15 1.24 5.16 4.93 1.32

d (11.6) s d (4.5) dd (4.5, 1.7) dd (11.6, 1.7) dd (8.5, 7.9) m m dd (6.8, 5.0) s s s s s

6.15 1.97 1.88 2.02

qq (7.2, 1.5) dq (7.2, 1.5) q (1.5) s

2.04 s 6.12 qq (7.2, 1.6) 1.98 dq (7.2, 1.6) 1.93 q (1.6)

6.14 qq (7.2, 1.4) 1.98 dq (7.2, 1.4) 1.92 q (1.4)

6.15 qq (7.2, 1.4) 1.95 dq (7.2, 1.4) 1.93 q (1.4)

6.17 2.00 1.93 3.36

qq (7.2, 1.4) dq (7.2, 1.4) q (1.4) s

6.09 qq (7.3, 1.3) 1.96 dq (7.3, 1.3) 1.83 q (1.3)

6.12 qq (7.2, 1.3) 1.99 dq (7.2, 1.3) 1.92 q (1.3)

3.21 s

Data were measured in methanol-d4. bData were measured in CDCl3.

to those of 19. In the HMBC spectrum (Supporting Information Figure S157) of 20, correlations of H-1/C-1′, H-10/C-1‴′, H-4/ C-1″, and H-5/C-1‴ indicated that the attachments of the two angeloyloxy and two acetoxy groups were at C-1 and C-10 and C-4 and C-5, respectively. The relative configuration of 20 was established by methods similar to those used for 16. As a result, the structure of 20 was assigned as shown. Pararubin U (21), a yellow oil, had a molecular formula of C24H36O10 on the basis of its HRESIMS and 13C NMR data, requiring seven indices of hydrogen deficiency. Comparison of the NMR data of 21 (Tables 2 and 4) and 20 revealed that they possessed a similar bisabolane-type sesquiterpenoid skeleton, except for the presence of only one angeloyloxy group in 21. Furthermore, the HMBC correlations (Supporting Information Figure S165) of H-4/C-1′, H-5/C-1″ and H-10/C-1‴ suggested that the two acetoxy groups and the angeloyloxy group of 21 were attached to C-4 and C-5, and C-10, respectively. Therefore, the structure of 21 was established as shown. Pararubin V (22), a yellow oil, has the molecular formula C24H34O9, as determined by the HRESIMS and 13C NMR data. The NMR spectroscopic data of 22 were similar to those of 21. However, according to the molecular formula, there were eight indices of hydrogen deficiency in 22, compared to seven for 21. The two acetoxy groups, an angeloyloxy group, a six-membered ring, and a terminal double bond accounted for seven of those, the remaining hydrogen deficiency was assigned to a furan ring formed between C-8 and C-11 on the basis of the 13C NMR chemical shifts and NOESY correlations (Supporting Information

Figure 4. Key HMBC (→), 1H−1H COSY (thick lines), and NOESY (↔) correlations of compound 16.

appropriate HMBC correlations. Therefore, the structure of 18 was determined as shown. Pararubin S (19), a yellow oil, has a molecular formula of C29H42O11 based on the HRESIMS and 13C NMR data, requiring ten indices of hydrogen deficiency. Comparison of its NMR spectroscopic data (Tables 2 and 4) with those of 16 revealed that 19 was the same type of bisabolane sesquiterpenoid with two angeloyloxy and two acetoxy groups, which were attached to C-1, C-8, and C-4 and C-5, respectively, according to the HMBC correlations (Supporting Information Figure S149). The methods applied for determining the relative configuration of 19 resembled those used for 16. Thus, the structure of 19 was determined as shown. Pararubin T (20) was isolated as a yellow oil, with a molecular formula of C29H42O11, based on the HRESIMS and 13C NMR data, requiring nine indices of hydrogen deficiency. The 1 H and 13C NMR data (Tables 2 and 4) showed the signal patterns characteristic of a bisabolane-type sesquiterpenoid similar G

DOI: 10.1021/acs.jnatprod.5b00380 J. Nat. Prod. XXXX, XXX, XXX−XXX

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341 polarimeter. UV spectra were recorded on a Jasco J-810 spectropolarimeter. IR spectra were recorded on a Bruker VERTEX 70 FT-IR microscopic spectroscopy. 1D and 2D NMR spectra were recorded on a Bruker-AM-400 spectrometer. HRESIMS spectra were obtained from a Thermo Scientific LTQ-Orbitrap XL mass spectrometer. Column chromatography was performed using silica gel (200−300 or 300−400 mesh; Qingdao Marine Chemical Inc.), Sephadex LH-20 gel (GE Healthcare), and MCI gel (CHP20P, 75−150 μm; Mitsubishi Chemical Industries Ltd.). HPLC was carried out on an Agilent 1260 system using YMC-Pack ODS-A HPLC column (250 mm × 10 mm i.d.) and YMC-Pack SIL-06 HPLC column (250 mm × 10 mm i.d.). Plant Material. P. rubescens were collected in the Lushan mountains of Jiangxi Province, People's Republic of China, in September 2012, and identified by research scientist Bei-Li Huang (Lushan Botanical Garden). A voucher specimen (ID 20120901) was deposited in the Herbarium of Materia Medica, Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, People's Republic of China. Extraction and Isolation. The air-dried whole plants of P. rubescens (18.4 kg) were extracted with 90% acetone (5 × 50 L) at room temperature and concentrated in vacuo to give 10 L of extract. The solution was sequentially partitioned with PE and EtOAc. The EtOAc-soluble fraction (180.0 g) was subjected to a silica gel column, eluting with PE/acetone (20:1−1:10 step gradient elution), to obtain five major fractions (A−E). Fraction B (62.3 g) was decolored on an MCI gel column (MeOH/H2O, from 75 to 100%) to afford three fractions, B1−B3. Fraction B1 was chromatographed on a Sephadex LH-20 column (MeOH) to give three subfractions (B1.1−B1.3). Fraction B1.1 was subjected to silica gel (PE/acetone, from 20:1 to 1:10), semipreparative HPLC (MeOH/H2O, 7:3), and normal-phase HPLC (n-hexane/2-propanol, 15:1) columns to yield 11 (11.7 mg) and 12 (8.7 mg). Fraction B1.2 was separated on ODS silica gel (MeOH/ H2O, from 30 to 100%), semipreparative HPLC (MeOH/H2O, 7:3), and normal-phase HPLC (n-hexane/2-propanol, 15:1) columns to yield 1 (15.5 mg), 2 (23.5 mg), 3 (17.3 mg), 4 (22.7 mg), 7 (4.3 mg), and 13 (3.7 mg), and similarly, fraction B1.3 was purified in the same way as B1.2 to yield 5 (3.1 mg), 6 (2.4 mg), 8 (2.8 mg), 9 (4.2 mg), and 10 (3.4 mg). Fraction B2 was fractionated using a Sephadex LH-20 column (MeOH) to produce three subfractions, B2.1−B2.3. Fraction B2.1 was further resolved by semipreparative HPLC (MeOH/H2O, 7:3) to afford 14 (4.9 mg) and 15 (1.6 mg). Fraction B2.2 was chromatographed on ODS silica gel (MeOH/H2O, from 30 to 100%), semipreparative HPLC (MeOH/H2O, 7:3), and normalphase HPLC (n-hexane/2-propanol, 15:1) columns to yield 16 (2.6 mg), 17 (3.3 mg), 18 (2.1 mg), 19 (15.0 mg), and 20 (4.8 mg). Fraction B2.3 was subjected to semipreparative HPLC (MeOH/H2O, 7:3) to give a subfraction, which was further purified by normal-phase HPLC (n-hexane/2-propanol, 15:1) to yield 21 (2.5 mg). Fraction B3 was chromatographed sequentially on Sephadex LH-20 (MeOH), ODS silica gel (MeOH/H2O, from 30 to 100%), semipreparative HPLC (MeOH/H2O, 7:3), and normal-phase HPLC (n-hexane/2-propanol, 15:1) columns to afford 22 (2.9 mg). Pararubin A (1). White needles (CH2Cl2); mp 134−136 °C; [α]25D −26 (c 0.14, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.37) nm; IR (KBr) νmax 3395, 1706, 1647 cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESIMS m/z 565.2605 [M + Na]+ (calcd for C27H42O11Na, 565.2625). Crystal data: C27H42O11, MW = 542.61, orthorhombic, space group P212121, a = 10.5266 (2) Å, b = 14.2874 (2) Å, c = 18.9078 (3) Å, α = β = γ = 90.00°, V = 2843.69(8) Å3, Z = 4, ρ (calcd) = 1.267 g·cm−3, and μ (Cu Kα) = 0.816 mm−1; reflections collected 18594, independent reflections 4884; final R indices [I > 2σ(I)] R1 = 0.0290, wR2 = 0.0743; R indices (all data) R = 0.0290, wR2 = 0.0744; largest diff peak and hole 0.298 and −0.377 eÅ−3; the absolute structure parameter 0.03 (13). Pararubin B (2). Colorless gum; [α]25D −32 (c 0.38, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.39) nm; IR (KBr) νmax 3428, 1705, 1648 cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESIMS m/z 565.2616 [M + Na]+ (calcd for C27H42O11Na, 565.2625).

Figure S174) of H-8/H-12. As a result, 22 appeared to be a bisabolane-type sesquiterpenoid possessing a two-ring system. The positions of the angeloyloxy and two acetoxy groups of 22 were confirmed by the HMBC correlations (Figure 5) of

Figure 5. Key HMBC (→), 1H−1H COSY (thick lines), and NOESY (↔) correlations of compound 22.

H-1/C-1′, H-4/C-1″, and H-5/C-1‴, and at C-1, C-4, and C-5, respectively. The relative configuration of 22 was established by methods similar to those used for 16. Therefore, the structure of 22 was deduced as shown. Compounds 1−22 belong to the highly oxygenated bisabolane-type sesquiterpenoids,12,13 some of which were reported to show varying degrees of antimicrobial and cytotoxic activities.14−18 Therefore, we examined the corresponding activities of compounds 1−22. The antimicrobial activities of compounds 1−22 were tested against Staphylococcus aureus, Escherichia coli, and Monilia albicans. However, only 13 and 20 showed weak antimicrobial activities against S. aureus and E. coli (Table 5). Table 5. Antibacterial Activity of Compounds 13 and 22a antibacterial activity (mm) sample

Staphylococcus aureus

Escherichia coli

13 22 penicillin G streptomycin

9 8 25

8 8 18

a

Antibacterial activity is measured by inhibitory zone diameter (IZD). Penicillin G and streptomycin were used as positive controls.

We next investigated all isolates for their in vitro cytotoxic activities against B16, HepG2, and MCF7 cell lines by the MTT assay with doxorubicin as a positive control. Compounds 13, 16, and 17 showed weak inhibitory activities against the three cell lines at the concentration of 100 μM (Supporting Information Table 1). The IC50 value of 13, 16, and 17 are respectively listed in Table 6. Table 6. Cytotoxicity of Compounds 13, 16, and 17 Against B16, HepG2, and MCF7 Cell Lines IC50 (μM)

a

compound

B16

HepG2

MCF7

13 16 17 doxorubicina

39.69 49.65 42.48 17.67

71.50 96.61 40.81 23.22

78.90 56.22 79.35 34.52

Doxorubicin was used as a positive control.

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EXPERIMENTAL SECTION

General Experimental Procedures. X-ray crystallographic analysis was collected on a Bruker APEX DUO diffractometer using Cu Kα radiation. Optical rotations were measured on a PerkinElmer H

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Pararubin C (3). Colorless gum; [α]25D −28 (c 0.34, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.37) nm; IR (KBr) νmax 3444, 1715, 1648 cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESIMS m/z 565.2617 [M + Na]+ (calcd for C27H42O11Na, 565.2625). Pararubin D (4). Colorless gum; [α]25D −17 (c 0.36, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.37) nm; IR (KBr) νmax 3416, 1718, 1648 cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESIMS m/z 565.2605 [M + Na]+ (calcd for C27H42O11Na, 565.2625). Pararubin E (5). Colorless gum; [α]25D −22 (c 0.07, CH3OH); UV (CH3OH) λmax (log ε) 217 (4.36) nm; IR (KBr) νmax 3376, 1704, 1646 cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESIMS m/z 565.2617 [M + Na]+ (calcd for C27H42O11Na, 565.2625). Pararubin F (6). Colorless gum; [α]25D −24 (c 0.11, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.35) nm; IR (KBr) νmax 3444, 1714, 1649 cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESIMS m/z 565.2619 [M + Na]+ (calcd for C27H42O11Na, 565.2625). Pararubin G (7). Colorless gum; [α]25D −18 (c 0.11, CH3OH); UV (CH3OH) λmax (log ε) 215 (4.33) nm; IR (KBr) νmax 3383, 1715, 1647 cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESIMS m/z 565.2618 [M + Na]+ (calcd for C27H42O11Na, 565.2625). Pararubin H (8). Colorless gum; [α]25D −17 (c 0.07, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.37) nm; IR (KBr) νmax 3411, 1715, 1647 cm−1; 1H and 13C NMR data see Tables 1 and 2; HRESIMS m/z 565.2617 [M + Na]+ (calcd for C27H42O11Na, 565.2625). Pararubin I (9). Colorless gum; [α]25D −10 (c 0.12, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.29) nm; IR (KBr) νmax 3465, 1715, 1648 cm−1; 1H and 13C NMR data see Tables 2 and 3; HRESIMS m/z 579.2761 [M + Na]+ (calcd for C28H44O11Na, 579.2781). Pararubin J (10). Colorless gum; [α]25D −17 (c 0.09, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.31) nm; IR (KBr) νmax 3407, 1703, 1647 cm−1; 1H and 13C NMR data see Tables 2 and 3; HRESIMS m/z 523.2514 [M + Na]+ (calcd for C25H40O10Na, 523.2519). Pararubin K (11). Colorless gum; [α]25D −22 (c 0.29, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.35) nm; IR (KBr) νmax 3406, 1697, 1646 cm−1; 1H and 13C NMR data see Tables 2 and 3; HRESIMS m/z 523.2499 [M + Na]+ (calcd for C25H40O10Na, 523.2519). Pararubin L (12). Colorless gum; [α]25D −35 (c 0.22, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.38) nm; IR (KBr) νmax 3417, 1699, 1647 cm−1; 1H and 13C NMR data see Tables 2 and 3; HRESIMS m/z 523.2514 [M + Na]+ (calcd for C25H40O10Na, 523.2519). Pararubin M (13). Colorless gum; [α]25D −29 (c 0.09, CH3OH); UV (CH3OH) λmax (log ε) 217 (4.52) nm; IR (KBr) νmax 3473, 1703, 1648 cm−1; 1H and 13C NMR data see Tables 2 and 3; HRESIMS m/z 601.2983 [M + Na]+ (calcd for C31H46O10Na, 601.2989). Pararubin N (14). Colorless gum; [α]25D −17 (c 0.12, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.49) nm; IR (KBr) νmax 3412, 1703, 1648 cm−1; 1H and 13C NMR data see Tables 2 and 3; HRESIMS m/z 521.2356 [M + Na]+ (calcd for C25H38O10Na, 521.2363). Pararubin O (15). Colorless gum; [α]25D −20 (c 0.12, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.30) nm; IR (KBr) νmax 3340, 1699, 1646 cm−1; 1H and 13C NMR data see Tables 2 and 3; HRESIMS m/z 521.2353 [M + Na]+ (calcd for C25H38O10Na, 521.2363). Pararubin P (16). Yellow oil; [α]25D −11 (c 0.07, CH3OH); UV (CH3OH) λmax (log ε) 218 (4.47) nm; IR (KBr) νmax 1749, 1720, 1647 cm−1; 1H and 13C NMR data see Tables 2 and 4; HRESIMS m/z 643.3090 [M + Na]+ (calcd for C33H48O11Na, 643.3094). Pararubin Q (17). Yellow oil; [α]25D −27 (c 0.08, CH3OH); UV (CH3OH) λmax (log ε) 217 (4.48) nm; IR (KBr) νmax 3507, 1749, 1721, 1649 cm−1; 1H and 13C NMR data see Tables 2 and 4; HRESIMS m/z 629.2935 [M + Na]+ (calcd for C32H46O11Na, 629.2938). Pararubin R (18). Yellow oil; [α]25D −17 (c 0.06, CH3OH); UV (CH3OH) λmax (log ε) 217 (4.42) nm; IR (KBr) νmax 1749, 1721, 1647 cm−1; 1H and 13C NMR data see Tables 2 and 4; HRESIMS m/z 643.3070 [M + Na]+ (calcd for C32H46O11Na, 643.3094). Pararubin S (19). Yellow oil; [α]25D −8 (c 0.38, CH3OH); UV (CH3OH) λmax (log ε) 217 (4.50) nm; IR (KBr) νmax 3510, 1719, 1648 cm−1; 1H and 13C NMR data see Tables 2 and 4; HRESIMS m/z 589.2619 [M + Na]+ (calcd for C29H42O11Na, 589.2625).

Pararubin T (20). Yellow oil; [α]25D −23 (c 0.12, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.36) nm; IR (KBr) νmax 3491, 1720, 1648 cm−1; 1H and 13C NMR data see Tables 2 and 4; HRESIMS m/z 589.2622 [M + Na]+ (calcd for C29H42O11Na, 589.2625). Pararubin U (21). Yellow oil; [α]25D −10 (c 0.06, CH3OH); UV (CH3OH) λmax (log ε) 217 (4.19) nm; IR (KBr) νmax 3418, 1743, 1719, 1647 cm−1; 1H and 13C NMR data see Tables 2 and 4; HRESIMS m/z 507.2201 [M + Na]+ (calcd for C24H36O10Na, 507.2206). Pararubin V (22). Yellow oil; [α]25D +11 (c 0.07, CH3OH); UV (CH3OH) λmax (log ε) 215 (4.16) nm; IR (KBr) νmax 1745, 1719, 1646 cm−1; 1H and 13C NMR data see Tables 2 and 4; HRESIMS m/z 489.2094 [M + Na]+ (calcd for C24H34O9Na, 489.2101). Antimicrobial Assays. The microbial strains of Staphylococcus aureus, Escherichia coli, and Monilia albicans were purchased from the Basic Medical College of Huazhong University of Science and Technology. The paper disk diffusion method19 was used as an antimicrobial test for compounds 1−22. About 0.1 mL of each sample (200 μg/mL) was added to a piece of paper (6 mm in diameter). Each disk was dried in air and pasted onto a culture dish at 37 °C for 24 h. Antibacterial activity was measured by the inhibitory zone diameter (IZD). Penicillin G, streptomycin, and fluconazole standard antimicrobial susceptibility disks (Hangzhou Microbial Reagent Co., Ltd.) were used as positive controls for S. aureus, E. coli, and M. albicans, respectively. Each test was performed in duplicate. Cell Viability Assay. The three cancer cell lines, including B16 (mouse melanoma cell lines), HepG2 (human hepatocellular carcinoma cell lines), and MCF7 (human breast adenocarcinoma cell lines), were purchased from the cell bank of the Basic Medical College of Huazhong University of Science and Technology. Each of these cell lines were maintained in DMEM containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2. Then 90 μL of the cell suspension (1 × 104 cell/mL) was seeded in each well of 96-well cell culture plates for 24 h. Then 10 μL of tested concentrations of compounds 1−22 were added to each well. After another 24 h of incubation, each well was treated with 15 μL of MTT solution (5 mg/mL) for 4 h at 37 °C. For MTT assay, the supernatant was discarded and 100 μL of DMSO was added to each well. The optical density at 570 nm was measured using a microplate reader. All samples were cultured in triplicate. The data (cell viability, measured by MTT assay) were normalized and expressed as a percentage of the control group, which is set to 100%.

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ASSOCIATED CONTENT

S Supporting Information *

Nomenclature, 1D and 2D NMR spectra, HRESIMS, IR, and cytotoxic activities of compounds 1−22 (PDF); crystallographic information file (CIF). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00380.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86 27 83657870. Fax: 86 27 83692739. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the staff at the analytical and testing center of Huazhong University of Science and Technology for collecting the spectroscopic data and Yan-song Peng from the Lushan Botanical Garden for taking the plant picture. This work was supported by the Natural Science Foundation of China (no. 31270394) and the Wuhan Program for Science and Technology (no. 2013060501010158). I

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DOI: 10.1021/acs.jnatprod.5b00380 J. Nat. Prod. XXXX, XXX, XXX−XXX