Polycyclic Polyprenylated Acylphloroglucinol Congeners from

Article pubs.acs.org/jnp

Polycyclic Polyprenylated Acylphloroglucinol Congeners from Hypericum scabrum Wan Gao,† Wei-Zhen Hou,† Jun Zhao,‡ Fang Xu,† Li Li,† Fang Xu,‡ Hua Sun,† Jian-Guo Xing,‡ Ying Peng,† Xiao-Liang Wang,† Teng-Fei Ji,*,† and Zheng-Yi Gu*,‡ †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Xinjiang Key Laboratory for Uighur Medicine, Institute of Materia Medica of Xinjiang, Urumqi 830004, People’s Republic of China S Supporting Information *

ABSTRACT: Twenty polycyclic polyprenylated acylphloroglucinols (PPAPs), including the new compounds hyperscabrones A−I (1−9), were isolated from the air-dried aerial parts of Hypericum scabrum. These compounds comprise seven different structural types. All structures were determined by NMR spectroscopic methods and both experimental and calculated electronic circular dichroism (ECD) spectra. The evaluation of their neuroprotective effects on glutamateinduced toxicity in SK-N-SH cells showed that compounds 4−7 exhibited significant neuroprotection at 10 μM. Additionally, compounds 3, 4, 7, and 9 showed moderate hepatoprotective activities against paracetamol-induced HepG2 cell damage at 10 μM.

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P

RESULTS AND DISCUSSION The air-dried aerial parts of H. scabrum were extracted with EtOH, and the extract was partitioned between H2O and petroleum ether. The petroleum ether layer was separated using silica gel columns and preparative TLC coupled with RP-18 silica gel HPLC; a total of 20 compounds with seven different scaffolds were obtained. The HRESIMS data (m/z 535.3046 [M + H]+) of hyperscabrone A (1) indicated a molecular formula of C33H42O6. The 13 C NMR data (Table 1) of 1 had a high degree of similarity to those of hyperibone G,9 except for the chemical shifts of C-23, -24, -25, and -26 (1: δC 86.2, 82.8, 19.7, and 19.4; hyperibone G: δC 90.3, 71.3, 26.9, and 24.1). Compared to hyperibone G, the 1H NMR spectrum of 1 displayed an additional signal (δH 7.65) and a shift for H-23 (Δδ + 0.33). The rest of the spectrum was similar, except for the small differences of the chemical shifts of protons of the tetrahydrofuran rings. These observations suggested that 1 and hyperibone G had similar structures and differed in the presence of a C-24 hydroperoxy group in 1 rather than a tertiary hydroxy group. The analysis of the HMBC and HSQC correlations also confirmed this deduction. The relative configuration of 1 was elucidated via the ROESY data. The ROESY correlation of H-23 (δH 4.97) and H-6b (δH 2.13) indicated that the orientation of the isopropyl group was α and the tetrahydrofuran moiety was formed between the C-5 isoprenyl side chain and the C-4 enolic hydroxy group. In

lants of the genus Hypericum (family Guttiferae), such as St. John′s wort, have been widely used as folk medicines because of their broad-spectrum antibacterial, antidepressant, and neuroprotective activities.1−4 A series of bioactive polycyclic polyprenylated acylphloroglucinols (PPAPs) have been obtained from the genus Hypericum.5 Thus, PPAPs have attracted attention of researchers in both chemistry and pharmacology. In 2015, approximately 300 PPAPs had been identified, the majority being isolated from the genus Hypericum.6 Hypericum scabrum belongs to the Hypericum genus of the Guttiferae family, which is rich in PPAPs;7−10 A decoction of the plant is used in traditional Chinese medicine to treat cancer.11 As part of a systematic search for natural PPAPs with novel structure and diverse bioactivities from plants in the genus Hypericum, 20 PPAPs were isolated from H. scabrum. The compounds obtained in the present study, including nine new compounds (1−9), comprise seven different structural types (see Figure 1). These diverse carbon skeletons are presumably biosynthesized from monocyclic polyprenylated acylphloroglucinols (MPAPs) by different cycloadditions, such as aldol condensations and Diels− Alder additions. The structures were elucidated by analysis of 1D and 2D NMR experiments and were subsequently confirmed by comparing with data of known compounds. The absolute configurations of the bicyclo[3.3.1]nonane-2,4,9-trione and monocyclic cores were defined by the electronic circular dichroism (ECD) exciton chirality method.12 Additionally, some of the compounds exhibited significant neuroprotective effects and hepatoprotective activities at 10 μM concentrations. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 5, 2015

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DOI: 10.1021/acs.jnatprod.5b01063 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of compounds 1−20 isolated from the aerial parts of H. scabrum.

H-6b (δH 2.09)/H-24 showed that the orientation of H-7 was α. Thus, the 2D structure of 2 was assigned as shown in Figure 3. Hyperscabrone C (3), a colorless oil, possessed a molecular formula C31H46O5 as established by its HRESIMS (m/z 499.3405 [M + H]+) data. The strong IR absorptions implied the presence of olefinic (1640, 1589 cm−1), carbonyl (1721 cm−1), and hydroxy (3487 cm−1) functionalities. Its 1H NMR spectrum contained signals of two olefinic protons of isoprenyl groups (δH 4.93, 1H, t, J = 6.8 Hz; 4.96, 1H, t, J = 6.0 Hz), eight methyls (δH 1.00, 1.22, 1.24, 1.24, 1.54, 1.66, 1.68, 1.72), and one 2-methylpropanoyl group (δH 1.10, 3H, d, J = 6.8 Hz; 1.17, 3H, t, J = 6.8 Hz; 2.52, 1H, m). The 13C NMR spectrum of 3 showed three carbonyl carbons (δC 211.5, 207.2, and 194.5) and four olefinic carbons (δC 133.6, 133.6, 121.9, and 121.8). Comparison of the 13C NMR data and molecular formula of 3 with those of oxepahyperforin13 indicated that 3 contained four fewer carbon atoms, including an olefinic moiety, suggesting that a methyl group in 3 replaced the C-1 isoprenyl group in oxepahyperforin.

addition, H-33 (δH 1.40) was correlated with H-27 (δH 2.24), and H-6a (δH 2.13) was correlated with H-7 (δH 1.75). According to these correlations, the β-orientation of the C-7 prenyl group was evident.5 Therefore, the relative configuration of 1 was determined as shown in Figure 2. The molecular formula of hyperscabrone B (2) was assigned as C30H36O4 from its 13C NMR and HRESIMS (m/z 461.2681 [M + H]+) data, 58 mass units less than that of hyperibone G.9 Analysis of the 1D and 2D NMR data (Table 1) implied that 2 was a PPAP derivative and that it shared the same benzoylphloroglucinol core as hyperibone G. Comparison of the 1D NMR spectroscopic data of 2 with those of hyperibone G revealed that the group [−C(CH3)2OH] linked to C-23 (δC 72.0) in hyperibone G was absent in 2. In addition, the HMBC and HSQC correlations confirmed the 2D structure of 2. The relative configuration of 2 was defined via a ROESY experiment. The correlations between H-29 (δH 1.40)/H-24 (δH 2.24) and B



Article

Table 1. 1H NMR and 13C NMR Spectroscopic Data for Compounds 1−4a (δ in ppm, J in Hz) 1 position

δC

1 2 3 4 5 6 7 8

78.2 193.2 115.8 171.9 59.6 39.1 42.3 47.7

9 10 11 12 13 14 15 16 17

204.8 192.9 136.6 127.9 127.8 131.8 127.8 127.6 21.9

18 19 20 21 22

120.1 133.3 25.7 17.7 30.7

23 24 25 26 27

86.4 82.8 19.7 19.4 25.5

28

122.1

1.31, s 1.29, s 2.24, m 1.74, m 4.98, br s

29 30 31 32 33

132.3 26.5 17.5 16.1 22.8

1.71, s 1.58, s 1.20, s 1.40, s

2 δH

2.13, dd (4.0, 13.2) 1.64, m 1.75, m

7.48, d (7.7) 7.20, t (7.7) 7.38, t (7.7) 7.20, t (7.7) 7.48, d (7.7) 3.15, dd (7.2, 13.2) 3.03, dd (7.2, 13.2) 5.05, t (7.4) 1.63, s 1.63, s 2.65, dd (10.4, 13.2) 1.93, dd (6.4, 13.2) 4.97, br s

δC 78.7 193.3 116.1 172.6 59.1 38.1 42.5 48.1 204.9 193.7 137.0 128.1 127.9 132.0 127.9 128.1 22.2 120.4 133.6 25.8 17.9 29.8 72.0 26.8 122.3 132.7 17.7

3 δH

δC

2.09, dd (3.6, 13.2) 1.60, m 1.72, m

7.46, d (8.0) 7.23, d (8.0) 7.38, t (7.4) 7.23, d (8.0) 7.46, d (8.0) 3.11, dd (7.2, 14.0) 3.01, dd (7.2, 14.0) 5.0, t (6.8) 1.64, s 1.62, s 2.78, q (12.4) 1.94, dd (5.6, 13.2) 4.68, t (9.2) 4.50, m 2.24, m; 1.77, m 4.98, t (6.0)

60.5 194.5 128.9 166.6 74.3 42.8 36.9 45.0

4 δH

2.05, m 2.06, m 1.40, m

δC 60.8 194.6 129.0 166.8 74.6 45.3 37.2 43.1

207.2 211.5 41.0 20.7 21.6 16.9 33.2 24.1 87.8

2.52 m 1.17, d (6.8) 1.10, d (6.8) 1.01, s 2.55, m; 2.04, m 1.84, m; 1.57, m 3.83, d (9.6)

207.4 210.9 48.0 17.7 24.4 11.7 17.2 33.5 26.9

72.6 25.9 25.9 26.7 121.9

1.24, s 1.24, s 2.07, m 4.95, br s

88.1 72.8 26.1 25.9 26.9

1.58, s

133.6 17.9 25.6 16.4 22.8

25.9

1.70, s

121.8

1.67, s 1.69, s 1.23, s 3.28, dd (15.2, 6.0) 3.15 dd (14.8, 6.0) 4.96, br s

23.1 16.4

1.40, s 1.22, s

133.6 18.1 25.5

1.55, s 1.73, s

122.1 133.8 18.1 25.7 16.7 23.0 122.2 133.6 18.3 25.7

δH

2.00, m 2.09, m 1.42, m

2.25, m 1.15, d (6.4) 1.70, m 0.86, t (7.6) 0.98, s 2.54, m; 1.92, m 1.81, m; 1.55, m 3.86, d (9.6) 1.24, s 1.23, s 2.04, m 4.94, t 1.66, s 1.67, s 1.24, s 3.30, dd (15.2, 6.0) 3.12, dd (15.2, 6.4) 4.93, t 1.54, s 1.72, s

a1 H NMR spectra measured at 400 MHz, 13C NMR spectra measured at 125 MHz; obtained in CDCl3. Assignments supported by the 2D NMR spectra.

Figure 2. Selected key HMBC and ROESY correlations for 1.


This deduction was confirmed by the key HMBC correlations from Me-26 (δH 1.23) to C-9 (δC 207.2), C-2 (δC 194.5), C-5 (δC 74.3), and C-6 (δC 42.8), as shown in Figure 4. In the ROESY spectrum of 3, H-17 (δH 3.83) was correlated with H-12 (δH

1.17), H-13 (δH 1.10), H-14 (δH 1.01), and H-15b (δH 2.55), and H-14 (δH 1.01) was correlated with H-21 (δH 2.07). In addition, the ROESY correlation between H-26 (δH 1.23) and H-14 (δH 1.01) was also evident. Those data implied that 3 and C



Article



1.21), and H-10a (δH 2.15) was correlated with H-13/14 (δH 1.06). According to these correlations, the α-orientation of the 2hydroxypropan-2-yl group at C-11 was evident. Additionally, the sign of the optical rotation of 6 is opposite to that of hyperibone I. Therefore, the relative configuration of hyperscabrone F (6) was assigned as shown in Figure 6. The molecular formula of hyperscabrone G (7) was determined as C32H48O5 on the basis of HRESIMS data (m/z 535.3375 [M + Na]+). Its IR absorptions implied the presence of hydroxy (3317 cm−1) and carbonyl functionalities (1777, 1741, and 1678 cm−1). The 1H NMR spectroscopic data of 7 showed the presence of a hydroxy proton [δH 7.83 (1H, brs)], three olefinic protons [δH 4.90 (1H, t, J = 6.5 Hz), 4.96 (1H, t, J = 7.0 Hz), and 5.02 (1H, t, J = 7.3 Hz)], one isobutyl group [δH 2.97 (1H, m), 1.48 (2H, m), 0.90 (3H, d, J = 6.5 Hz), 0.88 (3H, t, J = 6.5 Hz)], five methylenes, and eight methyl groups. The 13C NMR spectroscopic data of 7 showed three carbonyl carbons [δC 218.0, 210.1, and 206.9], six olefinic carbons [δC 136.3, 133.8, 131.9, 124.3, 122.2, and 116.0], an oxygenated tertiary carbon [δC 97.4], a dioxygenated secondary carbon [δC 108.0], 10 methyl carbons, six methylene carbons, two methine carbons, and three quaternary carbons. These data indicated that 7 was a prenylated phloroglucinol derivative. The 13C NMR spectroscopic data of 7 were similar to those of hyperibone J10 except for the chemical shifts of C-11−C-14. Compound 7 was presumed to be a C-1 substituted derivative of hyperibone J. In the HMBC spectrum, H-11 (δH 2.97) showed correlations with C-1 (δC 71.0), C-10 (δC 218.0), C-12 (δC 26.0), C-13 (δC 11.8), and C-14 (δC 16.5). This indicated that an isobutyl group located at C-1 of 7 replaced the isopropyl group in hyperibone J. The relative configurations of C-7 and C-8 were elucidated via the following ROESY results: HO-9 [δH 7.83 (1H, s)] with H-26 [δH 0.99 (3H, s)], H-26 with Hb-6 [δH 1.87 (1H, dd, J = 11.6, 2.8 Hz)], and Ha6 [δH 1.28 (1H, m)] with H-7 [δH 1.04 (1H, m)]. Therefore, the structure of hyperscabrone G (7) was assigned as shown in Figure 7. Hyperscabrone H (8) was obtained as a colorless oil. It showed a red spot on TLC plates when sprayed with the anisaldehydesulfuric acid reagent. The HRESIMS data of 8 exhibited a protonated molecule at m/z 363.3281 [M + H]+, consistent with a molecular formula of C23H38O3. Its IR spectrum showed an absorption peak at 1727 cm−1, suggesting the presence of a carbonyl functionality. Its 1H NMR spectroscopic data contained signals of eight methyl groups [δH 0.98 (s), 1.06 (d, J = 7.0 Hz), 1.08 (d, J = 7.0 Hz), 1.35 (s), 1.58 (s), 1.58 (s), 1.66 (s), 1.71 (s)], one methine [δH 4.68 (s)], and two olefinic protons [δH 4.98 (t, J = 7.0 Hz), 5.03 (t, J = 7.0 Hz)]. These data revealed the presence of two isoprenyl and one isopropyl group. The 2D structure of 8 was determined by analysis of its HMQC and HMBC data

oxepahyperforin had the same relative configurations at C-1, C-5, C-7, C-8, and C-17. Hyperscabrone D (4) was acquired as a colorless oil. The positive ion HRESIMS peak at m/z 513.3566 [M + H]+, in conjunction with 13C NMR data, revealed a molecular formula of C32H48O5, 14 mass units greater than that of compound 3. Compounds 4 and 3 had similar 1H and 13C NMR spectroscopic data except for the presence of a 2-methylbutanoyl group [(δH 0.86, 3H, t, J = 7.6 Hz; 1.15, 3H, d, J = 6.4 Hz; 1.70, 1H, m; 1.31, 1H, m; δC 210.9, 48.0, 24.4, 17.2, 11.7) in 4] instead of the 2methylpropanoyl group at C-5 in 3. These data implied that 4 was a PPAP derivative in which the C-10 isopropyl group of 3 was replaced by a s-butyl group, and the deduction was confirmed by HSQC and HMBC data. The same relative configurations of the C-1, C-5, C-6, C-7, and C-18 stereogenic centers in 4 and 3 were elucidated via the ROESY data. The relative configuration of C-11 was defined via the diagnostic ROESY correlation between H-11 (δH 2.25) and Me-15 (δH 0.98). Hyperscabrone E (5) was isolated as a colorless oil, and its HRESIMS data displayed a protonated molecule at m/z 501.2987 [M + H]+, indicating the molecular formula as C33H40O4. Its IR spectrum showed absorption bands that were consistent with carbonyl (1733 and 1672 cm−1) and aromatic (1629 cm−1) functionalities. The 13C NMR spectrum contained signals of three carbonyls (δC 206.4, 194.4, and 192.0), a benzene ring [δC 137.1, 128.8 ( × 2), 128.1 ( × 2), and 133.1], eight quaternary carbons, four methines, five methylenes, and seven methyls. The 1H NMR spectrum also indicated the presence of seven methyls and an aromatic ring. The assignments of 1H and 13 C NMR signals were confirmed by using HSQC and HMBC experiments. The 1D and 2D NMR data implied that 5 was a PPAP derivative that contained an aromatic ring. The 13C NMR spectrum of 5 resembled that of hyperibone I,9 with differences including the chemical shifts of C-23, −24, and −25. The HMBC correlations from C-23 (δC 87.9) to H-25a, 25b, and H-26 (δH 4.90, 4.87, and 1.59), and H-22a (δH 2.61) to C-4, C-5, C-6, and C-9 (δC 175.9, 58.7, 36.1, and 206.4) suggested that the tetrahydrofuran ring was formed between C-4 and C-5. The relative configurations of C-1, C-7, C-8, and C-23 were elucidated via the ROESY correlations shown in Figure 5. Hyperscabrone F (6) had a molecular formula of C33H42O5 based on HRESIMS data (m/z 519.3101 [M + H]+). Comparison of the 13C (Table 3) and 1H NMR (Table 2) spectroscopic data of 6 with those of hyperibone I9 revealed some small differences including the chemical shift of C-11 (6: δC 92.1; hyperibone I: δC 93.3). Thus, 6 was deduced to have the same skeleton as hyperibone I. In the ROESY spectrum of 6, H-10b (δH 2.68) was correlated with H-11 (δH 4.49) and H-33 (δH D



Article

Table 2. 1H NMR Spectroscopic Data for Compounds 5−8a (δ in ppm, J in Hz) position

5

6

7

8

9

δH

δH

δH

δH

δH

1 2 3 4 5 6 7 8 9 10

a1

2.40, m; 2.15, m

2.09, m

1.53, m

1.70, m

4.67, s

2.09, m 1.45, m 2.05, m

2.05, m 1.43, m 2.06, m 7.92, (OH)

2.58, m 1.08, d (7.0) 1.06, d (7.0)

2.42, m 1.04, d (6.8) 1.25, m 1.29, m 0.88, t (7.6) 0.98, s

1.87, dd (11.6, 2.8) 1.28, m 1.04, m 7.83, br s (HO-9)

2.69, dd (8.4, 13.6) 2.47, m

4.68, s

11 12

4.94, m

2.68, dd (10.0, 14.0) 2.15, dd (6.5, 14.0) 4.49, dd (6.4, 9.6)

13

1.59, s

1.06, s

0.89, t (6.0)

14 15

1.65, s

1.06, s

16 17 18

0.90, d (5.2) 2.78, dd (12.4, 6.4) 2.53, dd (12.4, 6.4) 5.03, t (6.0)

7.70, d (7.2) 7.36,t (7.8)

7.69, d (7.2) 7.39, t (7.3)

1.64, s

19

7.50, t (7.4)

7.53, t (7.2)

1.63, s

20 21

7.36, t (7.8) 7.70, d (7.2)

7.39, t (7.3) 7.69, d (7.2)

22 23 24 25

2.61, m 1.96, dd (5.6, 13.2) 5.13, dd (5.2, 11.2)

2.48, m 5.07, t (7.2)

1.14, s 2.08, m 1.62, m 4.91, t (5.2)

1.67, s

26 27

4.90, s 4.87, s 1.59, s 2.58, m

28

4.92, m

4.88, br s

29 30 31 32 33

1.70, s 1.68, s 1.20, s 1.06, s

1.67, s 1.60, s 1.11, s 1.21, s

2.97, m 1.48, m

1.55, s 1.69, s

1.63, s 2.41, m

0.99, s 1.64, m 1.48, m 2.08, m 1.87, m 4.98, m

1.58, s 1.71, s 2.09, m 2.03, m 5.03, m

1.62, m 1.49, m 2.08, m 1.86, m 4.98, m

1.58, s 1.70, s 2.08, m 2.03, m 5.03, m

1.58, s 1.66, s 1.35, s

1.59, s 1.66, s 1.34, s

0.99, s 1.73, m 1.60, m 2.26, m 1.81, m 4.97, t (5.6) 1.58, s 1.67, s

H NMR spectra measured at 400 MHz, obtained in CDCl3

(Figure 8) and comparison with hyperscabrin B14. The relative configurations of all stereogenic centers of 8 were determined by interpretation of ROESY correlations. Significant correlations (Figure 8) were observed between H-2 [δH 4.68 (s)], H-12 [δH 1.64 (m) and 1.48 (m)], and H-18 [δH 2.09 (m) and 2.03 (m)], and between H-5 [δH 2.05 (m)], H-11 [δH 0.99 (s)], and H-23 [δH 1.35 (s)]. Thus, the prenyl groups at C-3 and C-5 were αoriented, and the C-2 isobutyryl and C-6 methyl groups were βoriented. A difference of 14 atomic mass units between hyperscabrone I (9) and compound 8 was inferred from HRESIMS (m/z 399.2886 [M + Na]+) and 13C NMR data, establishing the

molecular formula of 9 as C24H40O3. Comparison of the 1D NMR data of 9 with those of 8 implied that their data were similar, except for the substituent at C-2. The HMBC correlations from H-8 [δH 2.42 (m)] to C-7 (δC 210.7), C-9 (δC 14.4), C-10 (δC 25.9), and C-11 (δC 11.6) confirmed that a 2methylbutanoyl group in 9 replaced the 2-methylpropanoyl group in 8. Similar ROESY correlations between 9 and 8 indicated that their relative configurations were identical. Thus, the structure of 9 was defined as shown. The 11 known compounds were identified as hyperibone J (10),10 8-hydroxyhyperforin 8,1-hemiacetal (11),13 scrobiculatone B (12),14 hypermongone G (13),12 hypermongone H E



Article

Table 3. 13C NMR Spectroscopic Data for Compounds 5−8a (δ in ppm, J in Hz) 5

6

7

8

9

position

δC

δC

δC

δC

δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

68.4 194.4 116.8 175.9 58.7 36.1 46.3 47.4 206.4 25.4 119.5 134.9 18.1 26.2 192.0 137.1 128.8 128.3 133.1 128.3 128.8 35.1 87.9 140.7 114.0 17.2 29.0 124.4 133.5 25.9 18.0 26.9 22.4

67.3 176.3 117.9 194.5 61.6 38.9 45.7 46.4 206.2 26.2 92.1 71.0 26.6 23.8 192.2 137.2 128.7 128.6 133.4 128.6 128.7 30.6 119.2 134.7 26.0 18.0 29.6 124.0 133.4 25.9 18.0 23.7 27.1

71.0 206.9 97.4 210.1 51.7 34.2 42.3 47.3 108.0 218.0 46.6 26.0 11.8 16.5 24.2 116.0 136.3 18.2 25.9 16.9 28.6 122.2 133.8 18.1 26.0 15.6 37.1 23.9 124.3 131.9 18.2 25.3

207.6 61.6 46.0 37.3 39.4 87.1 211.2 42.3 18.9 17.4 16.4 36.4 21.9 122.6 133.0 17.7 25.9 26.5 123.7 131.7 17.9 25.7 17.9

207.7 62.0 49.1 37.3 39.3 87.1 210.7 46.1 14.4 25.9 11.6 16.4 36.4 22.1 122.6 133.1 17.7 25.9 26.5 123.7 131.6 17.9 25.7 17.9



by the TDDFT method.16 Consequently, the overall pattern of the calculated ECD spectra of 1a, 4a, 5b, 7a, and 8b were consistent with the experimental data of 1, 4, 5, 7, and 8, respectively (Figure 9). The ECD spectra of compounds 2, 3, 6, and 9 (Figure 10) were similar to those of 1, 4, 1, and 8 implying that their absolute configurations were (1R, 5S, 7S)-2, (1R, 5S, 7S, 8R)-3, (1S, 5R, 7R, 11S)-6, and (2R, 3R, 5S, 6S)-9. Therefore, the absolute configurations of hyperscabrone A-I (1− 9) were defined as depicted. Generally, most of the known PPAPs isolated from H. scabrum formed a unique family of “diamond-like” caged metabolites that were most likely derived from 2,4,6-trihydroxybenzophenone (i) via a series of C-alkylations with dimethylallyl diphosphate (DMAPP).17 Compounds 3, 4, 7, 10, 13−15, and 17−18 possess a rare methyl substituent at C-1 or -5 instead of the prenyl or geranyl group observed in many other hyperforin analogues. Compounds 1, 2, 5, 6, and 13−18 possess a dihydrofuran ring that was most likely formed by cyclization of an enolic hydroxy group with a 3-methylbut-2-enyl side chain.12 Biosynthetically, 8, 9, and 19 are presumably formed from ii through elimination and cyclization reactions.15 Compound 20 is an adamantane-type PPAP with a “diamond-like” caged core. It may derive biosynthetically from the endo-BPAPs with a bicyclo[3.3.1]nonane core via the strongly nucleophilic enolic C-3 cyclizing onto C-27.6 The putative biosynthetic pathway toward the formation of 1−9 is shown in Scheme 1. Considering the fact that many PPAPs have been isolated previously and that these types of compouds have neuroprotective activities,6 compounds 1−20 were evaluated for neuroprotective effects on the glutamate-induced toxicity in SKN-SH cells. The result showed that compounds 4−7, 13, 15, 18, and 20 had significant neuroprotection at 10 μM (Table 4). Additionally, all compounds were also bioassayed for hepatoprotective activity against paracetamol-induced HepG2 cell damage, and the hepatoprotective activity drug bicyclol was used as the positive control.18 Compounds 3, 4, 7, 9−11, 14, 16, 19, and 20 exhibited moderate hepatoprotective activity (Table

a13

C NMR spectra measured at 125 MHz; obtained in CDCl3.

Figure 6. Selected ROESY correlations for 6.

(14),12 hyperibone A (15),9 hyperibone B (16),9 hypermongone C (17),12 hypermongone D (18),12 yezo’otogirin C (19),15 and hyperibone K (20),10 by comparison of their NMR and MS data with reported values. The absolute configurations of hyperscabrone A-I (1−9) were defined by experimental and calculated ECD spectra. The ECD spectra of five pairs of enantionmers (1a: 1R, 5S, 7S, 23R; 1b: 1S, 5R, 7R, 23S; 4a: 1R, 5S, 7S, 8R, 17S; 4b: 1S, 5R, 7R, 8S, 17R; 5a: 1R, 5S, 7S, 23S; 5b: 1S, 5R, 7R, 23R; 7a: 1R, 5R, 7S, 8S; 7b: 1S, 5S, 7R, 8R; 8a: 2S, 3S, 5R, 6R; 8b: 2R, 3R, 5S, 6S) were calculated F



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Figure 9. Calculated and experimental ECD spectra of 1 and 8.

Figure 10. Experimental ECD spectra of 1−9. (200−300 mesh), silica gel GF-254, silica gel H (Qingdao Haiyang Chemistry Company, Qingdao, China), and Sephadex LH-20 (40−70 μm; Healthcare Bio-Sciences AB, Uppsala, Sweden). Semipreparative HPLC was performed on an Agilent 1100 HPLC with a YMC-Pack ODS-A column (250 × 20 mm, 5 μm, Kyoto, Japan). See Table 6 for information about the cytotoxic effects of the new compounds. Plant Material. The aerial parts of H. scabrum were acquired from the Wusun Moutain, Xijiang Uygur Autonomous Region, People’s Republic of China, in August 2013. The plant was identified by Prof. Lin Ma. A voucher specimen (No. ID-S-2545) was deposited in the Institute of Materia Medica, Chinese Academy of Medical Sciences. Extraction and Isolation. The aerial parts of H. scabrum (15.0 kg, dry) were extracted with 95% EtOH (3 × 15.0 L) under reflux, and the extract (2.2 kg) was suspended in H2O (5.0 L) and then partitioned with petroleum ether (3 × 5.0 L) to yield the petroleum ether extract (750.0 g). The petroleum ether-soluble material (169.2 g) was fractionated using a silica gel column chromatography and eluted with petroleum ether-EtOAc (1:0 to 0:1) to afford nine fractions (Fr.A-I). Fr.B (35.9 g) was separated into nine subfractions (Fr.B1−B9) via silica gel H using petroleum ether-CH2Cl2 (20:1 to 1:1) as eluent. Fr.B3 was applied on a silica gel column eluted with petroleum ether-EtOAc (50:1 to 10:1) to yield 7 (788 mg). Fr.C was subsequently loaded onto a silica gel column using petroleum ether-acetone (50:1 to 0:100) as the eluent to give 17 subfractions (Fr.C1−C17). Fr.C1 was further purified by preparative HPLC (MeOH-H2O from 100:1 to 80:1) to afford 8 (7 mg), 5 (15 mg), 4 (51 mg), 3 (55 mg), 9 (4 mg), and 6 (11 mg). Similarly, compound 1 (36 mg) and compound 2 (10 mg) were isolated from Fr.E. Hyperscubrone A (1). Colorless oil; [α]20D −37 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.01), 247 (3.78) nm; ECD (MeOH) λ (Δε) 270 (−18.71), 306 (+1.21) nm; IR (KBr) υmax 3410, 2976, 2927, 1728,

5). The inhibitory effects of compounds 1−20 were examined against three human tumor cell lines Bel7402, HCT-8, and A-549 using the MTT method. However, compounds 1−20 did not show significant inhibitory activities. PPAPs are a class of promising molecules for drug development, especially the bicyclic polyprenylated acylphloroglucinols (BPAPs) and adamantane-type PPAPs. Some research data reported previously revealed that both hyperforin and adhyperforin exert inhibitory effects on the synaptosomal uptake of many neurotransmitters, such as L-glutamate, γ-aminobutyric acid (GABA), serotonin, norepinephrine, and dopamine, at low concentrations (IC50 = 2.0 μM). Additionally, the result of the current study also showed that hypericin compounds had moderate hepatoprotective activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 polarimeter (Jasco, Tokyo, Japan). UV spectra were recorded on a JASCO V-650 spectrophotometer (Jasco, Tokyo, Japan). ECD spectra were recorded on a JASCO J-810 spectrometer (Jasco, Tokyo, Japan). IR spectra were recorded on a Nicolet 5700 IR spectrometer (Thermo Nicolet, Waltham, MA, U.S.A.). NMR data were measured on a Varian Inova-500 spectrometer (Varian Inc., Palo Alto, CA, U.S.A.) and a Mecury-400 spectrometer (Varian Inc., Palo Alto, CA, U.S.A.) using TMS as an internal standard. HRESIMS spectra were collected using an Agilent 1100 LC/MSD TrapSL mass spectrometer (Agilent Technologies Ltd., Santa Clara, CA, U.S.A.). Column chromatography (CC) was carried out using silica gel G



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Scheme 1. Putative Biosynthetic Pathway toward the Formation of 1−9

1626, 1448, 1371 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 535.3046 [M + H]+ (calcd for C33H42O6, 535.3059). Hyperscubrone B (2). Colorless oil; [α]20D −39 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 246 (3.86), 275 (3.82) nm; ECD (MeOH) λ (Δε) 269 (−11.42), 307 (+0.91) nm; IR (KBr) υmax 2963, 2920, 1732, 1690, 1618, 1384 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 461.2681 [M + H]+ (calcd for C30H37O4, 461.2692).

Hyperscubrone C (3). Colorless oil; [α]20D −124 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.93), 252 (3.80) nm; ECD (MeOH) λ (Δε) 274 (+14.86), 308 (−16.39) nm; IR (KBr) υmax 3487, 2983, 2921, 1721, 1639, 1589, 1445, 1375 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 499.3405 [M + H]+ (calcd for C31H47O5, 499.3423). Hyperscubrone D (4). Colorless oil; [α]20D −75 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.93), 267 (3.69) nm; ECD (MeOH) λ (Δε) 265 (+14.28), 307 (−13.69) nm; IR (KBr) υmax 3506, 2973, 2930, 1724, H



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252 (+9.95), 298 (−5.44) nm; IR (KBr) υmax 2965, 2928, 1733, 1672, 1629, 1450, 1361 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 501.2987 [M + H]+ (calcd for C33H41O4, 501.3005). Hyperscubrone F (6). Colorless oil; [α]20D −18 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.89), 254 (3.70) nm; ECD (MeOH) λ (Δε) 264 (−29.55), 303 (−8.39) nm; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 519.3101 [M + H]+ (calculated for C33H43O5, 519.3110). Hyperscubrone G (7). Colorless oil; [α]20D + 7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.93), 284 (3.14) nm; ECD (MeOH) λ (Δε) 286 (+0.22), 321 (−0.21) nm; IR (KBr) υmax 3316, 2963, 2927, 1740, 1678, 1454, 1378 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 535.3375 [M + Na]+ (calcd for C32H48O5Na, 535.3399). Hyperscubrone H (8). Colorless oil; [α]20D + 8.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.78) nm; ECD (MeOH) λ (Δε) 297 (+3.70) nm; IR (KBr) υmax 3400, 2972, 2934, 1727, 1460, 1380 cm−1; 1 H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 363.3281 [M + H]+ (calcd for C23H39O3, 363.3276). Hyperscubrone I (9). Colorless oil; [α]20D + 24 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (3.78) nm; ECD (MeOH) λ (Δε) 296 (+1.03) nm; IR (KBr) υmax 3384, 2966, 2932, 1727, 1457, 1378 cm−1; 1 H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 399.2886 [M + Na]+ (calcd for C24H40O3Na, 399.2883). In Vitro Neuroprotection Bioassays. The neuroprotective effects of compounds 1−20 against glutamate-induced toxicity in SK-N-SH cells were evaluated with the MTT colorimetric method.19 In Vitro Hepatoprotection Bioassays. The hepatoprotective effects of compounds 1−20 were evaluated by the MTT colorimetric assay20 in HepG2 cells. Cytotoxicity Bioassays. The cytotoxic effects of compounds 1−20 at 10−5 M against Bel7402, A-549, and HCT-8 were determined using the MTT method.10

Table 4. Neuroprotective Effects of Selective Compounds (10 μM) Against Glutamate-Induced Toxicity in SK-N-SH Cellsa cell viability compound

(% of normal)

normal control resveratrol 4 5 6 7 13 15 18 20

100.0 ± 0.0 61.9 ± 0.9b 72.6 ± 1.5c 70.8 ± 0.5c 73.1 ± 3.7c 73.5 ± 2.9c 72.1 ± 2.3c 71.6 ± 4.7c 83.0 ± 3.6b 75.4 ± 4.2c 73.1 ± 1.8c

Results are expressed as the means ± SD (n = 3; for normal and control, n = 6); resveratrol was used as positive control (10 μM). bp < 0.001 vs normal. cp < 0.05 vs control.

a

Table 5. Hepatoprotective Effects of Selective Compounds (10 μM) Against Paracetamol-Induced HepG2 Cella cell viability

inhibition

compound

(% of normal)

(% of control)

normal control bicyclol 3 4 7 9 10 11 14 16 19 20

100.0 ± 1.2 29.4 ± 1.5 43.2 ± 2.4c 33.7 ± 1.1c 47.0 ± 5.4b 55.3 ± 2.1d 50.9 ± 3.6c 47.6 ± 2.1d 39.3 ± 3.8b 45.8 ± 1.9c 36.7 ± 2.9b 40.6 ± 5.3b 64.0 ± 8.0b

19.5 6.1 24.9 36.7 30.5 25.8 14.0 23.3 10.3 15.9 49.0

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01063. The original ESIMS, UV, IR, 1D NMR, 2D NMR, and ECD spectra for all new compounds and bioassay methods of all compounds (PDF)

Results are expressed as the means ± SD (n = 3; for normal and control, n = 6); bicyclol was used as positive control (10 μM). bp < 0.05. cp < 0.01. dp < 0.001.

a

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*E-mail for T.-F.J.: [email protected]. Tel.: +86-10-60212117. *E-mail for Z.-Y.G.: [email protected].

inhibitory rate compound

Bel7402

1 2 3 4 5 6 7 8 9 5-FUd

18.39 −1.32 19.45 8.64 −0.20 1.05 −3.16 14.68 3.31 70.57

AUTHOR INFORMATION

Corresponding Authors

Table 6. Cytotoxic Effects of New Compounds (10−5 M) Against Bel7402, A-549, and HCT-8 Using the MTT Method a

ASSOCIATED CONTENT

S Supporting Information *

Notes

HCT-8b

A-549c

−32.17 14.38 −37.08 −41.22 −29.72 3.04 −25.93 −25.72 −24.40 70.38

14.61 1.46 0.36 8.65 6.64 10.67 18.97 17.20 −13.63 68.32

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project was supported financially by the Municipal Twelfth Five-year Major projects of the People’s Republic of China (201130105-4). Hua Sun and Ying Peng are recognized for technical support in bioassays and Wen-Yi He is recognized for recording the NMR spectra.

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REFERENCES

(1) Josey, E. S.; Tackett, R. L. Int. J. Clin.Pharmacol. Ther. 1999, 37, 111. (2) Chatterjee, S. S.; Bhattacharya, S. K.; Wonnemann, M.; Singer, A.; Müller, W. E. Life Sci. 1998, 63, 499−510. (3) Schempp, C. M.; Pelz, K.; Wittmer, A.; Schöpf, E.; Simon, J. C. Lancet 1999, 353, 2129. (4) Marianna, D.; Emmanuel, A. T. Biomimetic Org. Synth. 2011, 433− 467. (5) Ciochina, R.; Grossman, R. B. Chem. Rev. 2006, 106, 3963−3986.

a

Human hepatoma bel cells. bHuman colon adenocarcinoma. cLung cancer. dPositive control.

1662, 1599, 1452, 1378 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 513.3566 [M + H]+ (calcd for C32H49O5, 513.3560). Hyperscubrone E (5). Colorless oil; [α]20D −10 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.93), 252 (3.31) nm; ECD (MeOH) λ (Δε) I



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Polycyclic Polyprenylated Acylphloroglucinol Congeners from

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