1,9-seco-Bicyclic Polyprenylated Acylphloroglucinols from Hypericum

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1,9-seco-Bicyclic Polyprenylated Acylphloroglucinols from Hypericum uralum Jing-Jing Zhang,†,‡,§ Xing-Wei Yang,†,§ Xia Liu,† Jun-Zeng Ma,† Yang Liao,†,‡ and Gang Xu*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Hyperuralones C−H (1−6), six new 1,9-secobicyclic polyprenylated acylphloroglucinols (1,9-seco-BPAPs) derived from the normal polyprenylated acylphloroglucinols with a bicyclo[3.3.1]nonane-2,4,9-trione core, together with six known analogues, were isolated from the aerial parts of Hypericum uralum. The structures of 1−6 were elucidated on the basis of the interpretation of NMR and MS spectroscopic data. The structure of attenuatumione B, a known compound isolated from H. attenuatum, was revised to that of a 1,9-seco-BPAP by NMR spectroscopic analysis and previous biomimetic synthesis methods. The inhibitory activities of these isolates on acetylcholinesterase were tested, and compounds 1 and 2 exhibited moderate activities with IC50 values of 9.6 and 7.1 μM, respectively. In a systematic search for new and bioactive natural PPAPs from Hypericum species,4,7 12 PPAPs including six new 1,9-secoBPAPs (hyperuralones C−H, 1−6) were characterized from Hypericum uralum Buch.-Ham. ex D. Don (Clusiaceae), a perennial shrub distributed mainly in Tibet and in the northwest of Yunnan Province, People’s Republic of China.8 There has been no phytochemical studies on this plant previously, except for the isolation of two caged PPAPs, hyperuralones A and B, by our group.9 Herein are described the isolation and structural elucidation of the new metabolites 1−6 and the inhibitory activity of all compounds obtained against acetylcholinesterase (AChE), as well as the structural revision of attenuatumione B. The MeOH extract of H. uralum was subjected to purification using silica gel column chromatography and preparative thin-layer chromatography followed by RP-18 silica gel HPLC and yielded six new 1,9-seco-BPAPs (1−6), together with six known compounds. The known compounds were identified as hyphenrones A, F, and D (7−9),4a hyperuralones A and B,9 and hypercohin A,7a respectively, by comparison of their spectroscopic data with literature values. Hyperuralone C (1) was obtained as a colorless gum. Its molecular formula was established from the 13C NMR and HRTOFMS data (m/z 591.3666, [M + Na]+) as C35H52O6, indicating 10 degrees of unsaturation. The 1H NMR spectrum (Table 1) exhibited five olefinic protons (δH 4.71, 4.92, 5.05, 5.65, and 5.71), an isopropyl group (δH 0.97, d; 1.10, d; 2.58, sept.; J = 6.8 Hz), and nine singlet methyls (δH 1.11−1.75). The 13C and DEPT spectra displayed 35 carbon resonances

Hypericum species accumulate polycyclic polyprenylated acylphloroglucinols (PPAPs) with diverse structures and bioactivities.1,2 PPAPs, featuring highly oxygenated and densely substituted acylphloroglucinol-derived cores decorated with prenyl or geranyl side chains, are a class of structurally fascinating and synthetically challenging natural products. They exhibit a broad range of biological activities including antiinflammatory, antibacterial, and cytotoxic effects, as well as central nervous system effects such as in antidepressant and memory-enhancing properties.1−3 About 300 compounds of the PPAP family have been reported up to now, of which the majority are bicyclic polyprenylated acylphloroglucinols (BPAPs) with a bicyclo[3.3.1]nonane-2,4,9-trione core, as exemplified by hyperforin.1 The basic carbon skeleton of these BPAPs was thought to be unmodified until the isolation of a series of 1,9-seco-BPAPs in a previous study.4 These 1,9seco-BPAPs are likely formed by cleavage of the C-1/C-9 bond of oxidated BPAPs via a retro-aldol mechanism and subsequent formation of a five-membered lactone moiety, as exemplified by hyphenrone A.4a Further cyclization of these substances involving aldol condensations or Diels−Alder addition may produce PPAPs with more complicated carbon skeletons, such as hyphenrones C and D.4a In fact, the first 1,9-seco-BPAP, perforatumone, was isolated from H. perforatum in 2004 and assigned an incorrect structure possessing a seven-membered carbon core.5 Later in 2014, the second 1,9-seco-BPAP, named attenuatumione B, was elucidated as an analogue of perforatumone, based on their closely matching 13C NMR data.6 However, the structure of perforatumone was proved to be incorrect and was further revised to hyphenrone A with an eight-membered carbon core, using NMR spectroscopic and biomimetic synthesis methods.4b © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 15, 2015

A

DOI: 10.1021/acs.jnatprod.5b00830 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

Table 1. 1H NMR Data for Compounds 1−6 (600 MHz, δ in ppm, J in Hz) 1a

no. 1 6

4.49, s 1.57, m

7 11 12 13

1.56, 2.58, 1.10, 0.97,

overlap sept (6.8) d (6.8) d (6.8)

2.97, 2.62, 4.92, 1.64, 1.64, 2.50, 2.08, 4.71, 1.59, 1.64, 2.15, 1.84, 5.05, 1.75, 1.64, 1.11, 2.27, 2.21, 5.65, 5.71, 1.33, 1.32,

dd (15.4, 6.8) m t (6.8) s s dd (15.4, 7.2) m t (7.2) s s m m brs s s s m m m d (15.4) s s

14 17 18 20 21 22 23 25 26 27 28 30 31 32 33 34 35 37 38 a

2a 4.46, 1.62, 1.58, 1.57, 2.41, 0.95, 1.70, 1.30, 0.90, 2.96, 2.63, 4.92, 1.65, 1.65, 2.51, 2.08, 4.71, 1.59, 1.63, 2.14, 1.83, 5.05, 1.75, 1.64, 1.12, 2.27,

s m m m m d (6.8) m m t (7.5) dd (15.0, 6.8) dd (15.0, 6.8) t (6.8) s s dd (15.0, 7.2) m t (7.2) s s m m brd (6.0) s s s m

5.64, 5.70, 1.33, 1.32,

m d (15.6) s s

3a

4a

4.49, s 1.58, overlap

4.47, s 1.60, overlap

1.54, 2.59, 1.10, 0.97,

1.57, 2.42, 0.95, 1.70, 1.31, 0.91, 2.97, 2.62, 4.92, 1.64, 1.64, 2.51, 2.07, 4.71, 1.60, 1.64, 2.15, 1.83, 5.07, 1.75, 1.64, 1.13, 2.30,

m m d (5.6) m m t (7.5) dd (13.8, 6.6) dd (13.8, 6.6) t (6.6) s s dd (15.0, 7.2) overlap t (7.2) s s m m brs s s s m

5.61, 5.72, 1.37, 1.37,

m d (15.6) s s

m sept (6.8) d (6.8) d (6.8)

2.94, m 2.64, m 4.92, t (6.9) 1.75, s 1.65, s 2.51, dd (15.1, 7.0) 2.05, m 4.71, t (7.0) 1.54, s 1.65, s 2.16, m 1.82,m 5.08, brs 1.65, s 1.65, s 1.13, s 2.19, m 2.25, m 5.64, m 5.72, d (15.6) 1.38, s 1.37, s

5b 4.33, 2.13, 1.78, 1.12, 2.57, 1.07, 1.03,

s overlap m m brs d (6.8) d (6.8)

6b

4.90, 1.63, 1.63, 2.64, 2.38, 4.80, 1.95, 1.68, 2.08, 1.80, 4.94, 1.74, 1.60, 1.17, 0.87,

m s s m m brs m s m m m s s s s

4.32, s 2.13, m 1.76, m 1.09, m 2.41, m 1.02, d (6.8) 1.70, m 1.33, m 0.81, t (7.2) 2.72, dd (14.7, 6.8) 2.65, overlap 4.88, t (6.8) 1.65, s 1.646, s 2.63, overlap 2.37, m 4.78, t (6.8) 1.95, m 1.68, s 2.09, m 1.79, m 4.94, brs 1.74, s 1.60, s 1.20, s 0.87, s

2.04, 5.02, 1.66, 1.58,

m t (6.8) s s

2.05, 5.02, 1.66, 1.59,

2.67, m

m t (6.8) s s

Recorded in acetone-d6. bRecorded in methanol-d4.

206.4 (C-10), one ester carbonyl at δC 172.1 (C-9), one oxygenated quaternary carbon at δC 95.6 (C-3), two quaternary carbons at δC 57.2 (C-5) and 49.0 (C-8), two methines at δC 63.5 (C-1) and 40.6 (C-7), and one methylene at δC 37.3 (C6), for a 1,9-seco-BPAP-type metabolite.4 Comparison of its 1D NMR data with those of hyphenrone A (7),4a a 1,9-seco-BPAP

(Table 2) attributable to seven quaternary carbons (including three carbonyls), three methines (including two olefinic ones), two methylenes, three methyls, and 19 other resonances assignable to one isobutyryl and three prenyl groups. Analysis of these data indicated the characteristic resonances of three nonconjugated carbonyls at δC 198.9 (C-2), 206.7 (C-4), and B

DOI: 10.1021/acs.jnatprod.5b00830 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Data for Compounds 1−6 (δ in ppm, 150 MHz)

a

no.

1a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

63.5, CH 198.9, C 95.6, C 206.7, C 57.2, C 37.3, CH2 40.6, CH 49.0, C 172.1, C 206.7 C 43.1, CH 19.8, CH3 19.0, CH3 30.5, 114.9, 139.0, 26.0, 18.1, 28.1, 118.7, 136.3, 25.9, 18.0, 29.2, 123.9, 134.9, 26.1, 18.2, 15.7, 40.6, 120.2, 144.6, 70.2, 30.5, 30.4,

CH2 C C CH3 CH3 CH2 C C CH3 CH3 CH2 C C CH3 CH3 CH3 CH2 CH CH C CH3 CH3

2a 63.8, 199.0, 95.5, 206.4, 57.3, 37.2, 40.5, 48.9, 172.1, 206.4, 49.7, 15.6, 27.0, 11.5, 30.5, 114.8, 139.0, 26.0, 18.1, 28.0, 118.7, 136.4, 26.0, 18.0, 29.2, 123.9, 134.9, 26.1, 18.2, 15.8, 40.5, 120.3, 144.5, 70.2, 30.5, 30.4,

CH C C C C CH2 CH C C C CH CH3 CH2 CH3 CH2 C C CH3 CH3 CH2 C C CH3 CH3 CH2 C C CH3 CH3 CH3 CH2 CH CH C CH3 CH3

3a 63.6, 198.9, 95.8, 206.9, 57.3, 37.3, 40.8, 48.9, 172.1, 206.9, 43.1, 19.8, 19.0,

4a

CH C C C C CH2 CH C C C CH CH3 CH3

30.6, CH2 114.8, C 139.1, C 26.0, CH3 18.2, CH3 28.2, CH2 118.7, C 136.4, C 25.9, CH3 18.0, CH3 29.2, CH2 123.9, C 134.9 C 26.0 CH3 18.1, CH3 15.7, CH3 40.8, CH2 124.1, CH 140.4, CH 81.5, C 25.1, CH3 24.9, CH3

64.0, CH 199.1, C 95.8, C 206.3, C 57.3, C 37.3 CH2 40.8 CH 48.8 C 172.1, C 206.3, C 49.7, CH 15.8, CH3 27.1, CH2 11.5, CH3 30.6, CH2 114.8, C 139.0, C 26.0, CH3 18.1, CH3 28.1, CH2 118.7, C 136.4, C 25.9, CH3 18.0, CH3 29.2, CH2 123.9, C 134.9, C 26.1, CH3 18.2, CH3 15.7, CH3 40.8, CH2 124.2, CH 140.3, CH 81.5, C 25.1, CH3 25.0, CH3

5b 67.4, 197.3, 94.3, 214.0, 58.9, 40.0, 48.0, 44.1, 173.9, 206.5, 45.3, 18.5, 17.9,

CH C C C C CH2 CH C C C CH CH3 CH3

32.0, 115.6, 140.0, 26.5, 18.3, 32.6, 118.8, 141.2, 41.3, 16.2, 30.6, 123.5, 135.2, 26.0, 18.3, 21.6, 20.6, 27.5, 124.8, 132.5, 26.0, 18.2,

CH2 CH C CH3 CH3 CH2 CH C CH2 CH3 CH2 CH C CH3 CH3 CH3 CH3 CH2 CH C CH3 CH3

6b 68.0, 197.1, 94.4, 214.1, 58.8, 40.1, 48.1, 43.9, 173.8, 205.8, 52.2, 15.5, 25.9, 11.7, 31.9, 115.6, 140.0, 26.1, 18.3, 32.7, 118.8, 141.2, 41.3, 16.2, 30.5, 123.5, 135.2, 26.1, 18.2, 21.6, 20.5, 27.5, 124.8, 132.5, 26.5, 17.9,

CH C C C C CH2 CH C C C CH CH3 CH2 CH3 CH2 CH C CH3 CH3 CH2 CH C CH2 CH3 CH2 CH C CH3 CH3 CH3 CH3 CH2 CH C CH3 CH3

Recorded in acetone-d6. bRecorded in methanol-d4.

from H. henryi, indicated that they are structurally similar. Instead of the olefinic quaternary carbon at δC 132.6 (C-36) and a methylene at δC 22.3 (C-34) in hyphenrone A, an oxygenated quaternary carbon at δC 70.2 and an olefinic methine (δC 120.2) appeared in 1, consistent with hydroxylation of C-36 and formation of a Δ34,35 double bond in 1. This inference was supported by the correlations of H2-33/H34/H-35 in the 1H−1H COSY spectrum, along with the HMBC correlations from Me-32 (δH 1.11, s) to C-33 (δC 40.6) and from both Me-37 (δH 1.33) and Me-38 (δH 1.32) to C-35 (δC 144.6) and C-36. The correlations of Me-32 with C-1, C-7, and C-8, of H-1 (δH 4.49) with C-2 and C-3, of H2-17 (δH 2.97 and 2.62) with C-2, C-3, and C-4, and of H2-22 (δH 2.50 and 2.08) with C-4, C-5, and C-6 in the HMBC spectrum (Figure 1) confirmed that its planar structure is the same as in 7. The cross-peaks of Me-32/H2-27, H-1/H-7, and H-1/H2-17 in the ROESY spectrum, conjugated with the five-membered lactone moiety in the molecular model, suggested that the relative configuration of 1 is the same as that of hyphenrone A. Thus, the structure of 1 (hyperuralone C) was elucidated as shown. The molecular formula of hyperuralone D (2) was determined as C36H54O6 by analysis of its 13C NMR (Table

Figure 1. Key HMBC, 1H−1H COSY, and ROESY correlations of 1.

2) and HRTOFMS data (m/z 605.3813, [M + Na]+), 14 mass units more than that of 1. Comparison of the 1H and 13C NMR spectroscopic data of 2 with those of 1 indicated that the isopropyl group in 1 is replaced by a sec-butyl group (C-11, δC 49.7; C-12, δC 15.6; C-13, δC 27.0; and C-14, δC 11.5) in 2. The correlations of Me-14 (δH 0.90) with C-11 and C-13 and of Me-12 (δH 0.95) with C-11 and C-10 (δC 206.4) in the HMBC spectrum were indicative of such a difference. Other parts of 2 were identical to those of 1 by analysis of the 2D NMR spectroscopic data. C

DOI: 10.1021/acs.jnatprod.5b00830 J. Nat. Prod. XXXX, XXX, XXX−XXX

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198.1, C-2; 95.3, C-3; 206.6, C-4; 56.9, C-5; 36.9, C-6; 40.1, C7; 48.5, C-8; and 171.9, C-9) in attenuatumione B were very closely comparable to those of compounds 1−4 and 7. Furthermore, the deshielded C-1 (δC 63.2) and H-1 (δH 4.38, s) resonances indicate that this methine functionality is more likely to be adjacent to two carbonyl groups (C-2 and C-10), rather than one carbonyl group in attenuatumione B. In addition, the correlation of H-17 (δH 2.85) with C-2 in the HMBC spectrum of attenuatumione B does not support its given structure. The evidence above indicated that the structure of attenuatumione B has also been assigned incorrectly. Hence, the supporting 1D and 2D NMR spectra (Figures S51−S53) supplied in the original paper were examined, and its backbone was determined to be the same as that of hyphenrone A. The HMBC correlation from Me-30 (δH 1.38) and Me-31 (δH 1.39) to C-28 (δC 120.8) and from H-28 (δH 5.60) to C-7 (δC 40.1) supported the initial assignment of a Δ27,28 double bond. However, the strong NOESY correlation between H-1 and H217 (δH 2.85 and 2.60) suggested the same orientation of H-1 and C-17. Accordingly, the structure of attenuatumione B is revised as shown in Figure 3.

Hyperuralones E (3) and F (4) shared the same carbon skeletons and relative configurations of 1 and 2, respectively, by analysis of their 1D (Tables 1 and 2) and 2D NMR spectroscopic data. The increased 16 mass units of the molecular weight (m/z 607.3629 and m/z 621.3758, [M + Na]+) observed in the HRTOFMS and 11.3 ppm downshift of C-36 (δC 81.5) in the 13C NMR spectra of 3 and 4 indicated that the hydroxy groups in 1 and 2 were replaced by a hydroperoxy group in 3 and 4, respectively. Hyperuralone G (5) was assigned the molecular formula C35H52O5 from its 13C NMR and HREIMS data. The 1H and 13 C NMR spectroscopic data of 5 (Tables 1 and 2) resembled those of hyphenrone A, especially the resonances of the eightmembered core and lactone moieties. In the HMBC spectrum of 5, the correlations of both the singlet Me-32 (δH 1.17) and Me-33 (δH 0.87) with C-1 (δC 67.4), C-7 (δC 48.0), and C-8 (δC 44.1) suggested that a gem-dimethyl (Me-32 and Me-33) is present at C-8. When compared to hyphenrone A, the absence of a prenylmethyl group at C-8 and the presence of a methylene at δC 41.3 (C-25) supported the occurrence of a geranyl group in 5, as evidenced by the 1H−1H COSY correlations of H2-22/ H-23 and H2-25/H2-34/H-35, together with the HMBC correlations from Me-26 (δH 1.68) to C-23 (δC 118.8), C-24 (δC 141.2), and C-25 and from both Me-37 (δH 1.66) and Me38 (δH 1.58) to C-35 (δC 124.8) and C-36 (δC 132.5) (Figure 2). The HMBC correlations of H2-22 with C-4 (δC 214.0), C-5

Figure 3. Structural revision of attenuatumione B and selected key HMBC correlations.

Alzheimer’s disease (AD) is a multifarious progressive neurodegenerative state among the elderly. Potentiation of central cholinergic activity by using acetylcholinesterase (AChE) inhibitors is considered as one of the major pharmacological means for the management of AD.10 Since some PPAPs are reported to be inhibitors of AChE,3 the inhibitory activities of the isolates were examined against AChE using the Ellman method.11 As a result, compounds 1 and 2 exhibited moderate AChE inhibitory activities with IC50 values of 9.6 and 7.1 μM, while the 10 other compounds did not show obvious activity.

Figure 2. Key HMBC, 1H−1H COSY, and ROESY correlations of 5.

(δC 58.9), and C-9 (δC 173.9) confirmed the connection of the geranyl group to C-5. In the ROESY spectrum, the cross-peaks of H-1/H2-27, H-28/H-23, and H-23/H-18 defined the αorientation of H-1, C-17, C-22, and C-27. Furthermore, the chemical shift of C-7 (δC 48.0) also supported the endo substituent of C-7 (δC 45−49 for endo and 41−44 for exo) according to the rule proposed by Ciochina and Grossman.1 In addition, the strong correlation of Me-26 (δH 1.68) with H2-22 demonstrated the E-configuration of the C-23/24 double bond in hyperuralone G (5). On the basis of analysis of its MS, 1D, and 2D NMR data (Tables 1 and 2), hyperuralone H (6) was shown to possess the same backbone and relative configuration as 5. The difference of 6 was the presence of a sec-butyl group at C-10 rather than an isopropyl group in 5. As mentioned above, two 1,9-seco-BPAPs were reported before the correct characterization of this type of PPAPs was made. In previous work, the structure of perforatumone was revised to hyphenrone A (7) by NMR spectroscopic and biomimetic synthesis methods.4b,5 The second compound, attenuatumione B, was isolated from H. attenuatum and characterized as an analogue of perforatumone on the basis of their matching 13C NMR data of the basic carbon skeleton.6 However, the chemical shifts of C-1 through C-9 (δC 63.2, C-1;

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV-2401PC spectrometer. IR spectra were recorded on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. 1D and 2D NMR spectra were recorded on a Bruker DRX-600 spectrometer using TMS as an internal standard. Unless otherwise specified, chemical shifts (δ) were expressed in ppm with reference to the solvent signals. ESIMS and HREIMS data were acquired on Waters Xevo TQS and Waters AutoSpec Premier P776 mass spectrometers, respectively. Semipreparative HPLC was performed on an Agilent 1100 HPLC with a Zorbax SB-C18 (9.4 × 250 mm) column. Silica gel (100−200 and 200−300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, People’s Republic of China), MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and Sephadex LH-20 (GE Healthcare) were used for column chromatography. Fractions were monitored by TLC (GF 254, Qingdao Marine D

DOI: 10.1021/acs.jnatprod.5b00830 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Note

reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E − S)/E × 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound).

Chemical Co., Ltd.), and spots were visualized by heating silica gel plates immersed in H2SO4 in ethanol. Plant Material. The aerial parts of H. uralum were collected in Linzhi village, Linzhi prefecture, Tibet, People’s Republic of China, in July 2011. The plant was identified by Dr. Zhi-Jian Yin, and a voucher specimen (20110724) has been deposited at the Kunming Institute of Botany. Extraction and Isolation. The aerial parts of H. uralum (21 kg) were powdered and percolated with MeOH (3 × 20 L) at room temperature for 24 h and filtered, and the solvent was evaporated in vacuo. The crude extract obtained (1.9 kg) was subjected to silica gel column chromatography eluted with CHCl3 to afford a PPAP-rich fraction (165 g). This fraction was separated on an MCI-gel column (MeOH−H2O, 75−100%) to produce six fractions (Fr. A−F). Fraction A (28 g) was chromatographed on a silica gel column, eluted with petroleum ether−EtOAc (from 1:2 to 0:1), to obtain seven fractions (Fr. A1−A7). Fr. A4 (5 g) was then separated on a Sephadex LH-20 column (CHCl3−MeOH, 1:1) to yield four fractions (A4a− A4d). Fr. A4a was was purified using silica gel (petroleum ether− EtOAc, from 100:1 to 0:1) to afford 3 (29 mg) and 4 (8 mg). Fr. A4b was further purified by preparative HPLC (MeCN−H2O, 9:1), combined with preparative TLC, to afford 1 (16 mg), 2 (11 mg), 5 (13 mg), 6 (4 mg), 7 (130 mg), 8 (25 mg), and 9 (14 mg). Similarly, hyperuralones A (8 mg) and B (7 mg) were obtained from fractions A3 (4.0 g) and D5 (3.8 g), and hypercohin A (12 mg) was obtained from fraction D6 (4.6 g), respectively. Hyperuralone C (1): colorless gum; [α]15D +3 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 202 (3.30) nm; IR (KBr) νmax 3438, 2972, 2930, 1813, 1760, 1734, 1629, 1451, 1383 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 591 [M + Na]+; HRTOFMS m/z 591.3666 (calcd for C35H52O6Na, 591.3661). Hyperuralone D (2): colorless gum; [α]15D +4 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 202 (3.40) nm; IR (KBr) νmax 3440, 2971, 2929, 1813, 1760, 1733, 1691, 1628, 1451, 1378 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 605 [M + Na]+; HRTOFMS m/z 605.3813 (calcd for C36H54O6Na, 605.3818). Hyperuralone E (3): colorless gum; [α]19D +2 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 203 (3.60) nm; IR (KBr) νmax 3432, 2975, 2929, 1813, 1759, 1734, 1710, 1630, 1453, 1382 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 607 [M + Na]+; HRTOFMS m/z 607.3629 (calcd for C35H52O7Na, 607.3610). Hyperuralone F (4): colorless gum; [α]19D +2 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 203 (3.40) nm; IR (KBr) νmax 3442, 2987, 2928, 1631, 1595, 1385, 1352 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 621 [M + Na]+; HRTOFMS m/z 621.3758 (calcd for C36H54O7Na, 621.3767). Hyperuralone G (5): colorless gum; [α]21D −37 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 202 (3.93) nm; IR (KBr) νmax 3440, 2970, 2923, 2854, 1811, 1757, 1739, 1693, 1634, 1447, 1382 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; positive ESIMS m/z 575 [M + Na]+; HREIMS m/z 552.3831 (calcd for C35H52O5, 552.3815). Hyperuralone H (6): colorless gum; [α]21D −38 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (3.99) nm; IR (KBr) νmax 3440, 2970, 2927, 2880, 2858, 1810, 1756, 1739, 1712, 1695, 1630, 1450, 1378 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 589 [M + Na]+; HREIMS m/z 566.3981 (calcd for C36H54O5, 566.3971). Acetylcholinesterase Inhibitory Assay. The acetylcholinesterase inhibitory activity of the compounds was assayed by the spectrophotometric method developed by Ellman et al.,11 with slight modifications. S-Acetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB, Ellman’s reagent), and acetylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical Co., Ltd. Compounds were dissolved in DMSO. The mixture containing 110 μL of phosphate buffer (pH 8.0), 10 μL of test compound (50 μM), and 40 μL of acetylcholinesterase (0.04 U/ 100 μL) was incubated for 20 min (30 °C). The reaction was initiated by the addition of 20 μL of DTNB (6.25 mM) and 20 μL of acetylthiocholine for AChE inhibitory activity, respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm after 30 min. Tacrine was used as positive control (IC50 0.33 μM). All the

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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.5b00830. The original MS, 1H and 13C NMR, HSQC, 1H−1H COSY, HMBC, and ROESY NMR spectra of compounds 1−6 and a copy of 1D and 2D NMR spectra of attenuatumione B (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: (86) 871-65217971. E-mail: [email protected]. cn. Author Contributions §

J. J. Zhang and X. W. Yang contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was financially supported by the Natural Sciences Foundation of Yunnan Province (No. 2015FA032), Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2015-ZZ07), the Foundation from Youth Innovation Promotion Association CAS to X.W.Y. and G.X., and the West Light Foundation of the Chinese Academy of Sciences to X.W.Y.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00830 J. Nat. Prod. XXXX, XXX, XXX−XXX