Methylated Polycyclic Polyprenylated Acylphloroglucinol Derivatives

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Cite This: J. Nat. Prod. 2018, 81, 2348−2356

Methylated Polycyclic Polyprenylated Acylphloroglucinol Derivatives from Hypericum ascyron Jia-Wen Hu, Meng-Jiao Shi, Jia-Jia Wang, Li Li, Jian-Dong Jiang,* and Teng-Fei Ji* 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

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S Supporting Information *

ABSTRACT: Hyperascyrins A−H (1−11) and four known compounds (12−15) were acquired from the air-dried aerial parts of Hypericum ascyron and were all identified as methylated polycyclic polyprenylated acylphloroglucinol derivatives. Their structures were established by NMR spectroscopy, experimental and calculated electronic circular dichroism (ECD) data, and comparison with established compounds. Compounds 8 and 9 showed protection against paracetamol-induced HepG2 cell damage at 10 μM. The neuroprotective activities of all compounds (10 μM) were evaluated, and compounds 1 and 8 exhibited mild neuroprotection against glutamate-induced toxicity in SK-N-SH cells. 204 (log ε 4.48) and 246 (log ε 4.19) nm indicated the presence of a benzoyl group, corresponding to the 1H NMR signals (δH 7.23, 7.38, and 7.48). Examination of its 1H NMR spectrum also revealed the presence of two olefinic protons (δH 5.01 and 5.08) and eight methyl groups (δH 1.14−1.72, s). The 13C NMR data contained signals for 34 carbons including three carbonyl carbons (δC 206.8, 193.9, and 188.2), two double bonds of the prenyl groups (δC 122.3, 124.6, 131.2, and 133.6), and five methylene groups. These data suggested that compound 1 was an analogue of uralodin B23 with a bicyclo[3.3.1]nonane core. A comparison of the NMR data of 1 and uralodin B indicated that an isoprenyl group in uralodin B was changed to a methyl group in 1 and revealed that the linkage site of the methyl group was C-5 (Δδ ca. 5.1 ppm). These deductions were confirmed by the cross-peak signals in the HSQC and HMBC spectra, especially signals from H3-22 (δH 1.38) to C-4 (δC 176.1), C-5 (δC 51.0), C-6 (δC 40.1), and C-9 (δC 206.8); from H2-17 (δH 2.03/1.50) to C-2 (δC 188.2), C-3 (δC 111.7), C-4 (δC 176.1), C-18 (δC 92.7), and C-19 (δC 72.1); and from H3-28 (δH 1.14) to C-1 (δC 79.7), C-7 (δC 43.3), C-8 (δC 48.9), and C-29 (δC 36.6), as shown in Figure 2. The C-7 chemical shift (δC 43.3) and the chemical shift difference between H-6α and H-6β (Δδ ca. 0.53) were in accordance with the classical reported rules.2,4 Combined with NOESY cross-peaks from H-6β (δH 1.50) to H-23a (δH 2.21), H-7 was established as α-oriented. The NOESY spectrum showed correlations from H3-28 (δH 1.14) to H-6β, H2-23 (δH

P

lants from the genus Hypericum, one genera of the family Guttiferae, have historically been used to treat disease in some areas of the world.1,2 It contains abundant polycyclic polyprenylated acylphloroglucinols (PPAPs), which possess diverse structures due to substitution with isopentenyl, geranyl, acyl, and even more highly substituted moieties.3−5 This class of compounds also exhibits extensive bioactivities, such as antidepressant, anti-inflammatory, antiviral, antibacterial, antitumor, antineurodegenerative, and anti-HIV activities.6−9 With complex structures, promising bioactivities, and challenging syntheses, PPAPs have become more and more attractive to researchers.10−15 Hypericum ascyron (family Guttiferae) has been used for the treatment of headache, hepatitis, trauma, rheumatism, and dysentery in China,16 and it has been a source of flavonoids,17 xanthones,18,19 and certain PPAPs.20−22 In the present study, 11 new PPAPs, hyperascyrins A−K (1−11), and four known PPAPs (12−15), all of which contain a bicyclo[3.3.1]nonane2,4,9-trione core, were obtained. They were all substituted by methyl and benzoyl groups. The isolation, elucidation of structures, and bioevaluation of these compounds are reported herein.

■

RESULTS AND DISCUSSION Hyperascyrin A (1) was isolated as a colorless oil, and its molecular formula was established as C34H44O5 according to the hydrogen adduct ion peak at m/z 533.3260[M + H]+ (calculated for C34H45O5, 533.3261), indicative of 13 indices of hydrogen deficiency. Absorption bands of hydroxy (3532 cm−1) and carbonyl groups (1725 and 1696 cm−1) were observed in the IR spectrum. The UV absorption maxima at © 2018 American Chemical Society and American Society of Pharmacognosy

Received: February 26, 2018 Published: October 31, 2018 2348

DOI: 10.1021/acs.jnatprod.8b00176 J. Nat. Prod. 2018, 81, 2348−2356

Journal of Natural Products

Article

Figure 1. Structures of compounds 1−15 isolated from the aerial parts of H. ascyron.

2.21 and 1.85), and H-12/16 (δH 7.48) indicating the same configuration at C-1, C-5, and C-8 as in uralodin B. H-17a (δH 3.07) and H-17b (δH 2.88) were both related with H-18 (δH 4.87), but only H-17b was related with H3-21 (δH 1.26) and H16 (Figure 3). Thus, H-18 was defined as α-oriented. Therefore, the structure of hyperascyrin A (1) was defined as shown in Figure 1. Hyperascyrin B (2) had the same molecular formula, C34H44O5, as hyperascyrin A (1) according to the HRESIMS (m/z 533.3259 [M + H]+, calculated for C34H45O5, 533.3261) and 13C NMR data. Comparison of the NMR data of 2 and 1 (Table 1) suggested that the 2D structure of 2 was identical to that of 1. Differences in their structures were established by NOESY experiments, which revealed the same orientations of

Figure 2. Selected HMBC correlations for 1 and 3.

Figure 3. Selected NOESY correlations for 1 and 2. 2349



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

79.7 188.2 117.1 176.1 51.0 40.1

7 8 9 10 11 12 13 14 15 16 17

43.3 48.9 206.8 193.9 137.1 128.0 127.9 132.0 127.9 128.0 27.6

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

92.7 72.1 24.8 23.9 15.3 27.4 122.3 133.6 25.9 18.0 13.8 36.6 25.3 124.6 131.2 25.7 17.7

δH, mult (J, Hz)

2.03, dd (13.6, 3.9); 1.50, m 1.83, m

7.48, 7.23, 7.38, 7.23, 7.48, 3.07, 2.88, 4.87,

d (7.6) t (7.6) t (7.6) t (7.6) d (7.6) dd (15.2, 10.7) dd (15.2, 6.9) dd (10.7, 6.9)

1.31, 1.26, 1.38, 2.21, 5.01,

s s s m; 1.85, m t (8.0)

1.72, 1.59, 1.14, 2.12, 2.24, 5.08,

s s s m; 1.53, m m; 1.97, m t (7.0)

1.64, s 1.62, s

2 δC 79.8 188.2 116.7 176.1 51.0 39.8 43.6 49.2 206.7 194.1 137.1 128.0 128.0 132.0 128.0 128.0 27.7 92.5 72.2 24.8 23.5 15.4 27.1 122.4 133.8 25.8 17.9 13.7 36.6 25.3 124.5 131.3 25.7 17.7

3

δH, mult (J, Hz)

2.00, dd (13.5, 3.7); 1.53, m 1.78, m

7.52, 7.29, 7.40, 7.29, 7.52, 3.02, 2.90, 4.87,

d (7.3) t (7.3) t (7.3) t (7.3) d (7.3) dd (15.2, 10.6) dd (15.2, 7.5) dd (10.5, 7.5)

1.29, 1.24, 1.38, 2.19, 4.97,

s s s m; 1.79, m t (7.0)

1.68, 1.58, 1.14, 2.12, 2.25, 5.09,

s s s m; 1.49, m m; 1.94, m t (7.0)

1.64, s 1.62, s

δC 80.1 190.0 120.1 179.3 51.3 40.3 43.0 49.0 206.1 193.6 136.6 128.2 128.1 132.2 128.1 128.2 70.5 93.5 71.9 27.1 26.6 14.8 27.3 122.1 133.8 26.0 18.0 13.9 36.7 25.1 124.4 131.4 25.7 17.7

δH, mult (J, Hz)

1.97, dd (13.7, 4.1); 1.54, m 1.80, m

7.53, 7.26, 7.40, 7.26, 7.53, 5.64, 3.43, 4.55, 3.76, 1.44, 1.43, 1.43, 2.18, 4.95,

d (7.4) m t (7.4) m d (7.4) dd (7.4, 2.5) d (2.5) (HO-17) d (7.4) s (HO-19) s s s m; 1.83, m t (7.0)

1.71, 1.59, 1.16, 2.04, 2.24, 5.06,

s s s m; 1.56, m m; 1.96, m t (6.2)

1.65, s 1.62, s

4 δC 79.7 189.4 119.9 179.1 51.3 40.3 43.3 49.8 206.3 193.8 137.1 127.9 128.1 132.1 128.1 127.9 71.4 92.8 72.0 27.6 26.0 15.3 27.1 122.2 133.8 25.8 17.9 13.8 36.7 25.2 124.5 131.4 25.7 17.8

δH, mult (J, Hz)

2.17, dd (13.5, 3.5); 1.58, m 2.04, m

7.50, 7.28, 7.41, 7.28, 7.50, 5.58, 3.55, 4.61, 3.59, 1.48, 1.45, 1.38, 2.21, 5.01,

m m t (7.4) m m dd (7.5, 2.4) d (2.4) (HO-17) d (7.5) s (HO-19) s s s m; 1.82. m t (7.0)

1.68, 1.58, 1.16, 2.13, 2.27, 5.08,

s s s m; 1.52, m m; 2.01, m t (7.0)

1.65, s 1.62, s

a1

H NMR and 13C NMR spectra were obtained in CDCl3 and measured at 400 and 125 MHz, respectively.Assignments are supported by the 2D NMR spectra.

Figure 4. Calculated and experimental ECD spectra of 1 and 2.

H-7, Me-22, and Me-28 in both structures. The β-orientation of H-18 in 2 was based on the correlations of H-17a (δH 3.02) to H-18 (δH 4.87) and H-16 (δH 7.52) and correlations of H321 (δH 1.24) to H-17b (δH 2.88) (Figure 3). Thus, the

structure of hyperascyrin B (2), a C-18 epimer of hyperascyrin A, was identified. The absolute configurations of the C-18 isomers 1 and 2 were defined via a comparison of the experimental and 2350



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Figure 5. Selected NOESY correlations for 3 and 4.

the dihydrofuran rings in compounds 5 and 6 were linked to C-2 and C-3 instead of C-3 and C-4 as in 1 and 2. In the NOESY spectra of 5 and 6, correlations were evident among H-6β, H3-28, and H2-23 (5: δH 1.41, 1.28, and 2.13/ 1.73; 6: δH 1.39, 1.24, and 2.15/1.75), as well as between H328 and H-12/16 (5: δH 1.28 and 7.56; 6: δH 1.24 and 7.58) that were identical to those in the NOESY spectrum of 1. Furthermore, H-12/16 (δH 7.56) was related to H3-20 (δH 0.88) in the spectrum of 5, indicating the α-orientation of H18. However, correlations between H-12/16 (δH 7.58) and H320 (δH 1.15) were absent in the spectrum of 6, but H-12/16 (δH 7.58) correlated to H-18 (δH 4.16), and H3-20 (δH 1.15) correlated to H-31 (δH 5.03). The main distinction between the compounds involved the chemical shifts of H-18, H3-20, H3-21, and C-19, which were in accordance with reported data.25 Therefore, compounds 5 and 6 were established as C18 epimers, as shown in Figure 1. Hyperascyrins G−I (7−9), colorless oils, all possessed a molecular formula of C34H44O6, 16 mass units greater than that of compound 5. The NMR data of 5−9 were significantly different as far as the chemical shifts of C-17 [δC 5 (26.4), 6 (26.6), 7 (69.9), 8 (70.5), and 9 (70.4)] were concerned. By considering the analyses of compounds 1−4, compounds 7−9 were speculated to be C-17-hydroxylated derivatives of compounds 5 and 6. This assumption was supported by the correlations from H-17 (δH 5.43) to C-2 (δC 174.4), C-3 (δC 122.3), C-4 (δC 190.7), and C-18 (δC 93.0). The relative configuration of the stereocenter of the core cyclohexanone ring was determined in the same way as for compounds 5 and 6. In the NOESY spectra of 7−9, correlations were evident among H-28, H-6β, H-23, and H12/16, the same as for the core cyclohexanone rings of 5 and 6. A NOESY cross-peak between H-18 and H-12/16 of 7 and 8 was absent, but it was present in the spectrum of 9. The correlations between H-20/21 (δH 7: 1.31/1.01; 8: 1.07/0.82) and H-12/16 (δH 7: 7.62; 8: 7.53) in the NOESY spectrum of 7 and 8 allowed the assignment of the β- and α-orientations for H-18 in compounds 9 and 7, 8, respectively. Furthermore, HO-17 [detected by 1H−1H COSY correlations between H-17 (δH 5.43) and HO-17 (δH 4.22)] of 7 and H-17 (δH 5.41) of 8 both correlated to H-12/16 and H-20/21, which showed the opposite orientation of their H-17. The NOESY correlation

calculated electronic circular dichroism (ECD) data as conducted by the time-dependent density functional theory (TDDFT) method24 (Figure 4). Thus, the (1R,5R,7S,8R,18R) and (1R,5R,7S,8R,18S) absolute configurations of 1 and 2, respectively, were defined. Hyperascyrins C and D (3, 4) both had a molecular formula of C34H44O6 as established by HRESIMS (3: m/z 549.3207 [M + H]+, calculated for C34H45O6, m/z 549.3211; 4: m/z 549.3208 [M + H]+, calculated for C34H45O6, m/z 549.3211) and 13C NMR data. The molecular formula showed an extra oxygen compared to 1 and 2 (C34H44O5). A more in-depth analysis of their NMR data showed that the structures of 3 and 4 resembled those of 1 and 2, except for the presence of a 17hydroxy group in 3 and 4. This was indicated by the deshielded chemical shifts of C-17 [δC 1 (δC 27.6), 2 (δC 27.7), 3 (δC 70.5), and 4 (δC 71.4)], as well as the HMBC cross-peaks from H-17 to C-2, C-3, C-4, C-18, and C-19. In the NOESY spectra of 3 and 4, correlations that determined the orientations of H-7, Me-22, and Me-28 were found to be in accordance with those of 1 and 2. NOESY correlations in 4 were observed between H-17 (δH 5.58) and H-12/16 (δH 7.53) and between H3-21 (δH 1.45) and H-6α (δH 2.17), indicating the β-orientation of the hydrogen at C-17 and C-18. However, the α-orientation of H-17 and H-18 of compound 3 was established via correlations with HO-17 (δH 3.43) and HO-19 (δH 3.76) with H-12/16 (δH 7.53) (Figure 5). The (1S,5R,7S,8R,17S,18R) and (1S,5R,7S,8R,17R,18S) of 3 and 4, respectively, were confirmed in a similar manner to those for compounds 1 and 2. The molecular formulas of hyperascyrins E and F (5 and 6) were confirmed as C34H44O5, in accordance with compound 1, via the protonated molecules at m/z 533.3260 [M + H]+ (calculated for C34H45O5, m/z 533.3261) and m/z 533.3262 [M + H]+ (calculated for C34H45O5, m/z 533.3261) in the HRESIMS spectra of 5 and 6, respectively. Comparative analyses of their NMR data (Table 2) revealed the commonality of a benzoyl group, a dihydrofuran ring, and two isoprenyl groups in their structures. C-2 and C-4 were confirmed by HMBC correlations from H3-22 to C-4 (5: δH 1.35 to δC 190.4; 6: δH 1.31 to δC 190.7) and from H3-28 to C2 (5: δH 1.28 to δC 172.0; 6: δH 1.24 δC 173.0) (Figure S1, Supporting Information). Their chemical shifts implied that 2351


Journal of Natural Products Table 2. 1H and

13

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C NMR Spectroscopic Data for Compounds 5−9a (δ in ppm, J in Hz) 5

position

δC

1 2 3 4 5 6

70.9 172.0 118.9 190.4 60.8 41.9

7 8 9 10 11 12 13 14 15 16 17

41.9 48.6 207.4 193.6 138.2 127.8 128.6 132.6 128.6 127.8 26.4

18 19 20 21 22 23

93.8 70.3 26.5 23.9 16.0 28.0

24 25 26 27 28 29

122.1 133.6 25.9 18.0 14.9 37.9

30

24.3

31 32 33 34

124.2 131.7 25.7 17.7

6

δH, mult (J, Hz)

2.04, dd (13.7, 4.3) 1.41, t (13.2) 1.85, m

7.56, d (7.7) 7.38, t (7.7) 7.51, t (7.7) 7.38, t (7.7) 7.56, d (7.7) 2.97, dd (13.4, 9.0) 2.91, dd (13.4, 9.0) 4.63, t (10.2) 0.88, 0.92, 1.35, 2.13, 1.78, 4.99,

s s s m m m

1.70, 1.58, 1.28, 2.03, 1.75, 2.13, 1.97, 5.01,

s s s m m m m m

1.67, s 1.60, s

δC 71.5 173.0 118.6 190.7 60.4 41.9 44.3 48.2 206.7 193.7 138.1 127.5 128.1 132.2 128.1 127.5 26.6 93.6 71.1 26.1 24.6 15.9 27.8 122.2 133.6 25.8 17.9 12.9 39.4 25.5 124.2 132.2 25.6 17.8

δH, mult (J, Hz)

1.99, dd (13.8, 4.0) 1.38, t (13.4) 1.76, m

7.58, d (7.4) 7.33, t (7.4) 7.46, t (7.4) 7.33, t (7.4) 7.58, d (7.4) 3.03, dd (14.9, 9.7) 2.85, dd (14.9, 10.3) 4.16, t (10.0) 1.15, 1.13, 1.31, 2.15, 1.75, 4.95,

s s s m m m

1.68, 1.56, 1.24, 1.95, 1.47, 2.33, 2.04, 5.03,

s s s m m m m t (6.7)

1.66, s 1.24, s

7 δC 70.7 174.4 122.3 190.7 61.2 41.8 41.9 48.8 206.9 193.4 138.0 128.0 128.4 132.6 128.4 128.0 69.9 93.0 72.0 27.5 25.2 16.0 28.0 121.9 133.8 25.9 18.0 14.9 37.9 24.3 124.0 131.9 25.7 17.8

8

δH, mult (J, Hz)

2.05, dd (13.8, 3.9) 1.43, t (13.0) 1.76, m

7.62, 7.38, 7.48, 7.38, 7.62, 5.43, 4.22, 4.32,

d (7.4) t (7.4) t (7.4) t (7.4) d (7.4) d (6.9) s (HO-17) d (6.9)

1.01, 1.31, 1.35, 2.11, 1.76, 4.96,

s s s m m m

1.69, 1.57, 1.30, 1.77, 1.69, 2.12, 1.86, 4.98,

s s s m m m m m

1.65, s 1.59, s

δC 71.2 174.3 122.0 191.5 60.9 42.2 41.7 49.5 207.2 193.4 138.1 128.6 128.0 132.8 128.0 128.6 70.5 100.4 70.1 26.4 23.6 15.6 28.0 121.5 133.8 25.9 18.0 14.8 38.0 24.4 124.2 131.7 25.7 17.8

9

δH, mult (J, Hz)

2.06, m 1.45, t (14.3) 2.05, m

7.53, 7.37, 7.50, 7.37, 7.53, 5.41,

d (7.2) t (7.2) t (7.2) t (7.2) d (7.2) d (5.0)

4.30, d (5.0) 0.82, 1.07, 1.35, 2.14, 1.79, 5.00,

s s s m m m

1.69, 1.58, 1.30, 1.78,

s s s m

2.16, m 1.99, m 5.02, m 1.67, s 1.60, s

δC 71.3 176.3 122.1 191.6 60.4 42.1 43.3 49.6 206.4 194.1 138.7 127.1 128.2 132.0 128.2 127.1 70.4 93.6 71.5 26.9 26.5 15.7 27.8 122.1 133.8 25.7 18.0 13.8 38.8 25.1 124.4 131.9 25.9 17.9

δH, mult (J, Hz)

2.05, m 1.47, t (12.8) 2.00, m

7.42, 7.30, 7.43, 7.30, 7.42, 5.37,

m m m m m dd(6.8, 2.9)

3.62, d (6.8) 1.08, 1.34, 1.33, 2.19, 1.75, 4.96,

s s s m m t (7.1)

1.66, 1.56, 1.34, 1.91, 1.74, 2.17,

s s s m m m

5.05, t (6.8) 1.67, s 1.62, s

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

Figure 6. Calculated and experimental ECD spectra of 3 and 4.

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Figure 7. Selected NOESY correlations for compounds 10 and 11.

Scheme 1. Putative Biosynthetic Pathway for Compounds 1, 3, and 10

dihydropyrane ring in 10. This conclusion was supported by the key HMBC correlations (Figure S1, Supporting Information). Based on similar NOESY correlations between 1 and 10, the β-orientation of Me-22 and Me-28 and α-orientation of H7 were assigned (Figure 7). In the NOESY spectrum collected in CDCl3, H-17b (δH 2.43) correlated to H3-21 (δH 1.47) and H-12/16 (δH 7.48), indicating that H3-21 and H-12/16 were cofacial. When the spectrum was collected in DMSO, a crosspeak was observed from HO-18 (δH 5.19) to H3-21 (δH 1.39), indicating the β-orientation of HO-18. Hyperascyrin H (11) was assigned a molecular formula of C34H44O5, which was in accordance with some of the abovementioned compounds on the basis of the HRESIMS (m/z 533.3261 [M + H]+) data. Distinct signals for a monosubstituted benzene moiety (δH 7.60, 7.46, and 7.30), two vinyl protons (δH 4.97 and 4.86), and eight methyl groups (δH 0.93−1.68, s) were displayed in the 1H NMR spectrum. Combined with the 13C NMR data for three carbonyl carbons (δC 206.8, 196.5, and 194.1) and an oxepane ring (δC 167.7,

between H-12/16 (δH 7.42) and H-17 (δH 5.37) of 9 was also detected, confirming the α-OH in both 8 and 9. Hyperascyrins E−I (5−9) are a group of compounds whose structures resembled those of hyperascyrins A−D (1−4). Therefore, the same experiments were conducted to define their absolute configurations as (1S,5R,7S,8R,18S) for 5, (1S,5R,7S,8R,18R) for 6, (1S,5R,7S,8R,17R,18S) for 7, (1S,5R,7S,8R,17S,18S) for 8, and (1S,5R,7S,8R,17S,18R) for 9 (Figure S3, Supporting Information). The molecular formula of hyperascyrin J (10) was established as C34H44O5, based on the observation of the hydrogen adduct iron at 533.3257 [M + H]+ (calculated for C34H45O5, 533.3261) in the HRESIMS spectrum. This characteristic molecular formula and the benzoyl group, two olefinic protons, and eight methyl groups shown in its 1H NMR data were similar to those of compound 1. However, discrepancies in the chemical shifts of C-4, C-18, and C-19 (δC 10: 168.7, 68.0, and 81.0, 1: 176.1, 92.7, and 72.1) indicated that the dihydrofuran ring in 1 was replaced by a 2353



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Table 3. 1H and 13C NMR Spectroscopic Data for Compounds 10 and 11a (δ in ppm, J in Hz)

71.8, 46.1, 33.5, 24.1, and 87.1), compound 11 was found to be structurally similar to uralione H26 except for the side chain at C-5. The HMBC experiments showed key correlations from H2-17 (δH 3.16/3.06) to C-2 (δC 167.7), C-3 (δC 126.7), and C-4 (δC 194.1); from H3-22 (δH 1.33) to C-4 (δC 194.1), C-5 (δC 61.2), C-6 (δC 42.6), and C-9 (δC 2 06.8); from H3-28 (δH 1.30) to C-1 (δC 71.8), C-7 (δC 36.8), C-8 (δC 46.1), and C-29 (δC 33.5); and from H2-30 (δH1.88/1.49) to C-29 (δC 33.5), C-31 (δC 87.1), and C-32 (δC 72.1) and demonstrated that this conjecture was valid and indicated a planar structure for 11 as shown in Figure 1. The relative configurations of the stereocenter of the cyclohexanone ring were in accordance with those of compound 1 based on similar NOESY correlations. The β-orientation of H-31 was determined via the NOESY correlations from H-31 to H3-28 and from H-31 to H-12/16. The (1R,5R,7S,8R,18S) and (1S,5R,7S,8R,31S) of 10 and 11, respectively, were ascertained via the experimental and calculated ECD data (Figure S4, Supporting Information). Based on comparison of their NMR and MS data with literature values27 the four known compounds (12−15) were defined as longistyliones A−D. These compounds are all carrying a methyl group at C-5, indicating that they are likely derived from 3-methyl-2,4,6trihydroxybenzophenone through a series of C-alkylations with dimethylallyl diphosphate28 (DMAPP). The dihydrofuran and dihydropyran rings in these compounds are most likely formed by the cyclization of an enolic hydroxy group with an isoprenyl side chain. However, HO-17 in compounds 3, 4, 7, 8, and 9 might have been derived from an oxidation reaction at the allylic position of the isoprenyl side chain before cyclization. Putative biosynthetic pathways for 1, 3, and 10 are shown in Scheme 1. Because many PPAPs exhibit neuroprotective effects,6,29 the compounds were assayed for their neuroprotective activities. The positive control group used resveratrol against glutamateinduced toxicity in SK-N-SH cells. However, only compounds 1 and 8 showed mild neuroprotective actives at 10 μM (Table 4). These compounds were evaluated for hepatoprotection against paracetamol-induced HepG2 cell damage. Compounds 8 and 9 showed obvious protection with improving cell viability 68.1% and 78.4%, respectively, compared with the control group, using bicyclol at 10 μM (Table 5). Previous studies of H. ascyron were mainly focused on polyprenylated spirocyclic acylphloroglucinol derivatives, PPAPs with spirocyclic carbon skeletons.20,21 However, we obtained a series of methylated PPAPs, possessing a bicyclo[3.3.1]nonane-2,4,9-trione core, suggesting that a different geographic environment might influence the secondary metabolites of the same species. It is notable that these compounds are diagnostically substituted with methyl and benzoyl groups, similar to that reported for compounds 12− 15.27

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10 position

δC

1 2 3 4 5 6

80.3 192.9 110.7 168.7 53.0 40.6

7 8 9 10 11 12 13 14 15 16 17

44.1 49.5 207.5 194.0 137.0 128.1 128 131.9 128 128.1 25.9

18 19 20 21 22 23

68.0 81.0 24.2 23.0 15.8 27.0

24 25 26 27 28 29

122.5 133.4 25.8 17.9 13.6 36.6

30

25.3

31 32 33 34

124.6 131.2 25.7 17.7

11

δH, mult (J, Hz)

δC

1.96, dd (13.7, 4.1) 1.47, overlap 1.73, m

7.48, 7.25, 7.37, 7.25, 7.48, 2.63, 2.43, 3.84,

m m t (7.4) m m dd (17.7, 4.6) dd (4.6, 3.5) dd (4.6, 3.5)

1.27, 1.47, 1.34, 2.18, 1.81, 4.95,

s s s m m t (7.7)

1.68, 1.58, 1.15, 2.13, 1.43, 2.26, 1.96, 5.08,

s s s m m m m t (6.4)

1.64, s 1.62, s

71.8 167.7 126.7 194.1 61.2 42.6 36.8 46.1 206.8 196.5 137.1 128.4 128.1 132.6 128.1 128.4 22.7 120.7 133.1 25.6 18.0 16.5 27.0 121.9 133.6 25.8 17.9 17.4 33.5 24.1 87.1 72.1 25.6 24.8

δH, mult (J, Hz)

2.05, dd (13.8, 4.7) 1.53, m 2.17, m

7.60, 7.30, 7.46, 7.30, 7.60, 3.16, 3.06, 4.86,

d (7.4) t (7.8) t (7.4) t (7.8) d (7.4) dd (14.5, 6.9) dd (14.5, 6.3) t (6.6)

1.64, 1.66, 1.33, 2.12, 1.70, 4.97,

s s s m m t (7.1)

1.68, 1.57, 1.30, 2.38, 2.01, 1.88, 1.49, 3.72,

s s s t (13.5) m m m d (9.1)

0.94, s 0.93, s

a1

H NMR and 13C NMR spectra were obtained in CDCl3 and measured at 400 and 125 MHz, respectively. Assignments are supported by the 2D NMR spectra.

Table 4. Evaluation of Selected Compounds (10 μM) for Neuroprotective Effects against Glutamate-Induced Toxicity in SK-N-SH Cellsa

EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotation values were recorded with a JASCO P-2000 polarimeter (Jasco, Tokyo, Japan). The UV spectra were obtained using a JASCO V-650 spectrophotometer (Jasco, Tokyo, Japan). The ECD spectra were obtained using a JASCO J-810 spectrometer (Jasco, Tokyo, Japan). The IR spectra were obtained using a Nicolet 5700 IR spectrometer (Thermo Nicolet, Waltham, MA, USA). The NMR spectra were recorded using a Mecury-400 spectrometer (Varian Inc., Palo Alto, CA, USA) and a Varian Inova-500 spectrometer using CDCl3 as

compound

cell viability (% of normal)

normal control resveratrolb 1 6 7 8

100.0 ± 0.0 67.7 ± 1.1c 82.5 ± 1.2d 82.9 ± 8.7d 71.8 ± 2.1d 72.2 ± 1.6d 78.2 ± 0.5d

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

a

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Hyperascyrin C (3): colorless oil; [α]20D −67 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 204 (4.52), 249 (4.22) nm; ECD (MeOH) λ (Δε) 244.5 (+7.88), 207.0 (−22.19) nm; IR (KBr) νmax 3426, 2977, 2925, 1727, 1696, 1619, 1452, 1382 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 549.3207 [M + H]+(calcd for C34H45O6, 549.3211). Hyperascyrin D (4): colorless oil; [α]20D +3 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 204 (4.45), 248 (4.21) nm; ECD (MeOH) λ (Δε) 247.0 (+13.29), 271.0 (−9.01) nm; IR (KBr) νmax 3432, 2976, 2927, 1725, 1697, 1620, 1455, 1407, 1383 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 549.3208 [M + H]+ (calcd for C34H45O6, 549.3211). Hyperascyrin E (5): colorless oil; [α]20D +16 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.32), 248 (4.01) nm; ECD (MeOH) λ (Δε) 215.5 (−9.77), 249.5 (+12.37) nm; IR (KBr) νmax 3571, 2975, 1723, 1698, 1654, 1623, 1448, 1386 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 533.3260 [M + H]+(calcd for C34H45O5, 533.3261). Hyperascyrin F (6): colorless oil; [α]20D −48 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 205 (4.39), 248 (4.14) nm; ECD (MeOH) λ (Δε) 217.5 (−9.16), 250.5 (+9.72) nm; IR (KBr) νmax 3562, 2976, 2933, 1711, 1696, 1650, 1625, 1446, 1400, 1375 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 533.3262 [M + H]+(calcd for C34H45O5, 533.3261). Hyperascyrin G (7): colorless oil; [α]20D +12 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 204 (4.43), 253 (4.20) nm; ECD (MeOH) λ (Δε) 215.5 (−12.24), 247.0 (+19.76) nm; IR (KBr) νmax 3375, 2977, 2929, 1726, 1656, 1613, 1448, 1385 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 571.3026 [M + Na]+ (calcd for C34H44O6Na, 571.3030). Hyperascyrin H (8): colorless oil; [α]20D +28 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 204 (4.44), 251 (4.17) nm; ECD (MeOH) λ (Δε) 217.0 (−16.16), 248.5 (+15.12) nm; IR (KBr) νmax 3571, 3430, 2975, 2926, 1724, 1649, 1621, 1448, 1387 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 571.3027 [M + Na]+ (calcd for C34H44O6Na, 571.3030). Hyperascyrin I (9): colorless oil; [α]20D −35 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.41), 250 (4.16) nm; ECD (MeOH) λ (Δε) 219.5 (−16.00), 248.0 (+5.70) nm; IR (KBr) νmax 3374, 2976, 2931, 1726, 1657, 1613, 1457, 1386 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 571.3027 [M + Na]+ (calcd for C34H44O6Na, 571.3030). Hyperascyrin J (10): colorless oil; [α]20D −68 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 204 (4.39), 248 (4.13) nm; ECD (MeOH) λ (Δε) 247.0 (+5.80), 272.5 (−15.79) nm; IR (KBr) νmax 3503, 2977, 2925, 1723, 1695, 1603, 1450, 1383 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 533.3257 [M + H]+ (calcd for C34H45O5, 533.3261). Hyperascyrin K (11): colorless oil; [α]20D −201 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (4.47), 249 (4.25) nm; ECD (MeOH) λ (Δε) 222.5 (−16.22), 247.0 (+6.08) nm; IR (KBr) νmax 3493, 2975, 2934, 1722, 1692, 1652, 1595, 1448, 1378 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 533.3261 [M + Na]+ (calcd for C34H45O5, 533.3261). Neuroprotection Bioassays. All isolates were evaluated for in vitro neuroprotective activitiy against glutamate-induced toxicity in SK-N-SH cells with the MTT assay.30 Hepatoprotection Bioassays. All isolates were evaluated for in vitro hepatoprotective effects against paracetamol-induced HepG2 cell damage with the MTT assay.31

Table 5. Evaluation of Selected Compounds (10 μM) for Hepatoprotective Effects against Paracetamol-Induced HepG2 Cella compound

cell viability (% of normal)

inhibition (% of control)

normal control bicyclolb 4 8 9 11 14

100 ± 1.4 29.9 ± 6.7c 45.6 ± 0.4e 39.1 ± 1.5d 50.3 ± 1.3e 51.2 ± 1.4e 41.0 ± 0.2d 42.7 ± 0.9d

52.6 30.8 68.1 71.4 37.2 42.9

Results are expressed as the means ± SD (n = 3; for normal and control, n = 6). bPositive control (10 μM). cp < 0.001 vs normal. dp < 0.05. ep < 0.01 vs control.

a

solvent (δC 77.0 and δH 7.26). The HRESIMS spectra were measured via an Agilent 1100 LC/MSD TrapSL mass spectrometer (Agilent Technologies Ltd., Santa Clara, CA, USA). Semipreparative HPLC was conducted with an Agilent 1100 HPLC using a YMC-Pack ODSA column (20 × 250 mm). Column chromatography (CC) was performed with Sephadex LH-20 (40−70 μm; Healthcare BioSciences AB, Uppsala, Sweden), silica gel H, silica gel GF-254, silica gel (200−300 mesh) (Qingdao Haiyang Chemistry Company, Qingdao, China), and MCI gel CHP20P (35−75 μm, Mitsubishi Chemical Corp. Japan). Plant Material. The air-dried aerial parts of H. ascyron were purchased from Bozhou, Anhui Province, People’s Republic of China, in November 2013. Prof. Lin Ma was responsible for the identification of the plant. A voucher specimen (No. ID-S-2527) was deposited in the Institute of Materia Medica, Chinese Academy of Medical Sciences. Extraction and Isolation. The air-dried aerial parts of H. ascyron (75.0 kg) were extracted using reflux with 95% EtOH (750 L × 3), and the crude extract was suspended in H2O (15 L) and partitioned with petroleum ether (15 L × 3) to obtain a petroleum ether extract (998 g). A portion (711 g) of the extract was fractionated by CC over silica gel eluting with petroleum ether/EtOAc (1:0 to 0:1) to give nine fractions (Fr.A−I). Fr.G (84.2 g) was separated into nine fractions (Fr.Ga−Gi) on an MCI-gel column eluting with MeOH/ H2O (7:3 to 10:0). Fr.Gf (12.3 g) was loaded onto a silica gel column and eluted with petroleum ether/EtOAc (6:1 to 3:1) to yield eight fractions (Fr.Gf-1−Gf-8). Fr.Gf-4 was purified on a silica gel column by elution with petroleum ether/EtOAc (6:1), while Fr.Ge-7 was loaded onto a silica gel column and eluted with CH2Cl2/EtOAc (20:1). The fractions were subjected to semipreparative HPLC (MeOH/H2O) to give 5 (20 mg) and 1 (15 mg). Ten fractions (Gd1−Gd-10) were obtained from Fr.Gd via a silica gel column eluted with petroleum ether/EtOAc (6:1 to 3:1) and were purified by silica gel CC eluted with CH2Cl2/EtOAc 15:1, 20:1, or 30:1 and finally yielding 2 (99 mg), 3 (12 mg), 4 (30 mg), 6 (143 mg), 7 (5 mg), 8 (8 mg), 9 (14 mg), and 11 (29 mg) by semipreparative HPLC (MeOH/ H2O, 80−100%). Similarly, compounds 10 (5 mg), 12 (110 mg), 13 (166 mg), 14 (36 mg), and 15 (86 mg) were obtained from Fr.H (29.7 g). Hyperascyrin A (1): colorless oil; [α]20D −80 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.48), 246 (4.19) nm; ECD (MeOH) λ (Δε) 247.0 (+6.54), 279.0 (−11.90) nm; IR (KBr) νmax 3532, 2976, 2927, 1725, 1696, 1451, 1382 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 533.3260 [M + H]+ (calcd for C34H45O5, 533.3261). Hyperascyrin B (2): colorless oil; [α]20D −5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 206 (4.34), 247 (4.15) nm; ECD (MeOH) λ (Δε) 247.5 (+9.10), 277.5 (−5.84) nm; IR (KBr) νmax 3411, 2978, 2935, 1725, 1697, 1615, 1381 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 533.3259 [M + H]+ (calcd for C34H45O5, 533.3261).

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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.8b00176. 2355



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Article

(20) Hashida, W.; Tanaka, N.; Kashiwada, Y.; Sekiya, M.; Ikeshiro, Y.; Takaishi, Y. Phytochemistry 2008, 69, 2225−2230. (21) Zhu, H.; Chen, C.; Liu, J.; Sun, B.; Wei, G.; Li, Y.; Zhang, J.; Yao, G.; Luo, Z.; Xue, Y. Phytochemistry 2015, 115, 222−230. (22) Kong, L. M.; Long, X. W.; Yang, X. W.; Xia, F.; Khan, A.; Yan, H.; Deng, J.; Li, X.; Xu, G. Tetrahedron Lett. 2017, 58, 2113−2117. (23) Chen, X. Q.; Li, Y.; Cheng, X.; Wang, K.; He, J.; Pan, Z. H.; Li, M. M.; Peng, L. Y.; Xu, G.; Zhao, Q. S. Chem. Biodiversity 2010, 7, 196−204. (24) Bringmann, G.; Bruhn, T.; Maksimenka, K.; Hemberger, Y. Eur. J. Org. Chem. 2009, 2009, 2717−2727. (25) Yang, X. W.; Yang, J.; Xu, G. J. Nat. Prod. 2017, 80, 108−113. (26) Zhou, Z. B.; Li, Z. R.; Wang, X. B.; Luo, J. G.; Kong, L. Y. J. Nat. Prod. 2016, 79, 1231−1240. (27) Cao, X.; Yang, X.; Wang, P.; Liang, Y.; Liu, F.; Tuerhong, M.; Jin, D. Q.; Xu, J.; Lee, D.; Ohizumi, Y. Bioorg. Chem. 2017, 75, 139− 148. (28) Gao, W.; Hu, J. W.; Xu, F.; Wei, C. J.; Shi, M. J.; Zhao, J.; Wang, J. J.; Zhen, B.; Ji, T. F.; Xing, J. G. Fitoterapia 2016, 115, 128− 134. (29) Gao, W.; Hu, J. W.; Hou, W. Z.; Xu, F.; Zhao, J.; Sun, H.; Xing, J. G.; Peng, Y.; Wang, X. L.; Ji, T. F. Tetrahedron Lett. 2016, 57, 2244−2248. (30) Lei, H.; Zhao, C. Y.; Wang, X. L.; Peng, Y. J. Asian Nat. Prod. Res. 2014, 16, 854−864. (31) Hu, J.-W.; Gao, W.; Xu, F.; Wei, C.-J.; Shi, M.-J.; Sun, H.; Zhen, B.; Wang, J.-J.; Ji, T.-F.; Jiang, J.-D. Bioorg. Med. Chem. Lett. 2017, 27, 4932−4936.

The original ECD spectra, NMR, IR, UV, and HRESIMS for the new compounds, as well as detailed experiments of bioassays for all compounds (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J. D. Jiang). Tel: +86-1063017906. *E-mail: [email protected] (T. F. Ji). Tel: +86-10-63165226. ORCID

Teng-Fei Ji: 0000-0001-6168-3325 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project was financially supported by Projects of International Cooperation and Exchanges NSFC (NSFC-VR, No. 81361138020), the CAMS Innovation Fund for Medical Sciences (CIFMS), and the CAMS Initiative for Innovative Medicine (CAMS-I2M, No. 2016-I2M-1-010). W.-Y. He, X.-J. Jin, Y. Peng, and H. Sun are recognized for collecting the NMR spectra and support for the bioassays.

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Methylated Polycyclic Polyprenylated Acylphloroglucinol Derivatives

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