Nodulisporiviridins A–H, Bioactive Viridins from Nodulisporium sp

May 15, 2015 - ... Chinese Medicine and Natural Products, College of Pharmacy, .... Silvana Tommasini , Carmela Cannavà , Christian Celia , Massimo F...
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Nodulisporiviridins A−H, Bioactive Viridins from Nodulisporium sp. Qin Zhao,†,∥ Guo-Dong Chen,†,∥ Xiao-Lin Feng,† Yang Yu,† Rong-Rong He,† Xiao-Xia Li,† Yan Huang,‡ Wen-Xia Zhou,‡ Liang-Dong Guo,§ Yi-Zhi Zheng,⊥ Xin-Sheng Yao,† and Hao Gao*,† †

Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China ‡ State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, People’s Republic of China § State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ⊥ Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, People’s Republic of China S Supporting Information *

ABSTRACT: Eight new viridins, nodulisporiviridins A−H (1−8), were isolated from the extract of an endolichenic fungal strain Nodulisporium sp. (No. 65-17-2-1) that was fermented with potatodextrose broth. The structures were determined using spectroscopic and X-ray crystallographic analysis. Nodulisporiviridins A−D (1−4) are unique viridins with an opened ring A. The Aβ42 aggregation inhibitory activities of 1−8 were evaluated using a thioflavin T (ThT) assay with epigallocatechin gallate (EGCG) as the positive control (EGCG IC50 of 0.5 μM). Nodulisporiviridin G (7) displayed potent inhibitory activity with an IC50 value of 1.2 μM, and the preliminary trend of activity of these viridins as Aβ42 aggregation inhibitors was proposed. The short-term memory assay on an Aβ transgenic drosophila model of Alzheimer’s disease showed that all eight compounds improved the short-term memory capacity, with potencies close to that of the positive control (memantine). control (EGCG).14 That work was the first report of viridins’ activity against Aβ42 aggregation. The fungus (No. 65-12-7-1) produced additional analogues when it was grown in potato-dextrose broth (PDB). The EtOAc extract of the PDB fermented material was chemically investigated, and eight new viridins (nodulisporiviridins A−H (1−8)) were isolated. Notably, nodulisporiviridins A−D (1−4) possess unique viridin frameworks with ring A opened. In the ThT assay, nodulisporiviridin G (7) had the most potent activity (IC50 = 1.2 μM) among these eight new viridins. To further investigate the anti-AD effects of these compounds, short-term memory enhancement activities were evaluated using a human Aβ42 transgenic drosophila model. The assay showed that all eight compounds improved the short-term memory capacity of the AD flies, with activities close to that of the positive control (memantine). Details of the structure characterization, anti-Aβ42 aggregation activities, and short-term memory enhancement activities of 1−8 are reported herein.

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lzheimer’s disease (AD) is an age-related, progressive neurodegenerative disorder, which is characterized by the deposition of the amyloid-β peptide (Aβ) in the form of senile plaques.1 According to the amyloid hypothesis, the accumulation of Aβ initiates a neurodegenerative cascade that leads to progressive decline in learning, memory, and other cognitive functions.1 Therefore, prevention of Aβ aggregation is a promising strategy to prevent and treat AD.2,3 To date, many natural products have been found to possess anti-Aβ42 aggregation activity, including polyphenols (curcumin,4 epigallocatechin gallate (EGCG),5 procyanidins,6 cryptotanshinione,7 caffeoylquinic acids,8 isobavachalcone,9 brazilin,10 (+)-taxifolin,11 and catechins12), polysaccharides (glucan LJW0F213), steroids (demethoxyviridin14), and diphenyl ethers (violaceols15). In our previous chemical investigation of an endolichenic fungus Nodulisporium sp. (No. 65-12-7-1) that was grown using rice, nodulisporisteroids A and B (the first 3,4-seco-4-methyl progesteroids) and demethoxyviridin (a known viridin) were isolated. Demethoxyviridin exhibited potent anti-Aβ42 aggregation activity in a dose-dependent manner with the thioflavin T (ThT) assay, with an IC50 value close to that of the positive © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 16, 2014

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DOI: 10.1021/np500912t J. Nat. Prod. XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION Compound 1 was obtained as a colorless crystal plate. The quasi-molecular ion at m/z 343.1175 [M + H]+ by HRESIMS indicated the molecular formula of 1 was C19H18O6 (11 degrees of unsaturation). The 13C NMR spectrum showed 19 carbon signals, which corresponded to the molecular formula. Combined with the DEPT experiment, these carbons can be categorized as two carbonyls (δC 205.6 and 173.2), 10 aromatic or olefinic carbons [including three sp2 methine carbons (δC 146.7, 127.1, and 126.8)], one oxygenated sp3 quaternary carbon (δC 69.3), one oxygenated sp3 methine carbon (δC 61.5), four sp3 methylene carbons [including an oxygenated carbon (δC 58.1)], and one methyl carbon (δC 32.6). The 1H NMR spectrum also revealed that 1 had three olefinic or aromatic protons [δH 8.06 (1H, br s), 7.99 (1H, d, J = 8.1 Hz), and 7.93 (1H, d, J = 8.1 Hz)] and one methyl [δH 1.77 (3H, s)]. All proton resonances were associated with the directly attached carbon atoms in the HSQC experiment. The analysis of the 1H−1H COSY experiment and the coupling values of the protons revealed the presence of three isolated spin systems (C-1−C-2−C-3, C-11−C-12, and C-15−C-16), as shown in Figure 1a. Combined with the 1H−1H COSY data, the HMBC

elucidated as (S)-7-((S)-1,3-dihydroxypropyl)-6-hydroxy-6methyl-1H-cyclopenta[7,8]naphtho[2,3-b]furan-3,10(2H,6H)dione and named nodulisporiviridin A. Compound 2 was obtained as a yellowish, amorphous powder. The quasi-molecular ion at m/z 327.1235 [M + H]+ by HRESIMS indicated that the molecular formula of 2 was C19H18O5 (11 degrees of unsaturation). The 13C NMR spectrum of 2 showed 19 carbons, which corresponded to the molecular formula. Combined with the DEPT experiment, these carbons can be categorized as two carbonyls (δC 205.6 and 173.1), 10 aromatic or olefinic carbons [including three sp2 methine carbons (δC 146.3, 127.1, and 126.7)], one oxygenated sp3 quaternary carbon (δC 69.1), five sp3 methylene carbons [including an oxygenated carbon (δC 60.3)], and one methyl carbon (δC 31.7). The 1H NMR spectrum also showed that 2 had three olefinic or aromatic protons [δH 7.99 (1H, d, J = 8.1 Hz), 7.94 (1H, br s), and 7.93 (1H, d, J = 8.1 Hz)] and one methyl [δH 1.68 (3H, s)]. The nonexchangeable proton resonances were associated with the directly attached carbon atoms in the HSQC experiment. Except for the loss of an oxygenated methine carbon (δC 61.5) and the appearance of an additional methylene carbon (δC 19.9), the NMR data of 2 were similar to those of 1, which indicated that 2 and 1 had the same skeleton, and 2 was a dehydroxylated derivative of 1. The key 1H−1H COSY correlations between H2-2 [δH 1.83 (2H, m)] and H2-1 [δH 3.51 (2H, m)]/H2-3 [δH 2.75 (2H, m)] and the HMBC cross-peaks from H2-3 to C-4/C-20 determined that dehydroxylation occurred at the C-3 position. The planar structure of 2 was established by analyzing the 2D NMR data (HSQC, 1H−1H COSY, and HMBC) (Figures S13−S15, Supporting Information), which confirmed the above deduction. The assignments of all proton and carbon resonances are shown in Table 1. In the ECD experiment, 2 and 1 had similar ECD curves (Figure 3), which suggested that 2 shared an identical configuration at C-10 with 1. Therefore, the absolute configuration of 2 was assigned as 10S, and the structure of 2 was elucidated as (S)-6-hydroxy-7-(3-hydroxypropyl)-6-methyl1H-cyclopenta[7,8]naphtho[2,3-b]furan-3,10(2H,6H)-dione and named nodulisporiviridin B. Compound 3 was obtained as a yellowish, amorphous powder. The molecular formula was established as C18H16O5 (11 degrees of unsaturation) by the quasi-molecular ion at m/z 313.1074 [M + H]+ in the HRESIMS, which was 14 atomic mass units fewer than 2. The 13C NMR spectrum of 3 showed 18 carbons, which corresponded to the molecular formula. Combined with the DEPT experiment, these carbons can be categorized as two carbonyls (δC 205.6 and 173.0), 10 aromatic or olefinic carbons [including three sp2 methine carbons (δC 146.9, 127.0, and 127.0)], one oxygenated sp3 quaternary carbon (δC 69.1), four sp3 methylene carbons [including an oxygenated carbon (δC 60.4)], and one methyl carbon (δC 32.0). The 1H NMR spectrum also showed that 3 had three olefinic or aromatic protons [δH 7.99 (1H, d, J = 8.1 Hz), 7.95 (1H, br s), and 7.93 (1H, d, J = 8.1 Hz)] and one methyl [δH 1.68 (3H, s)]. Except for one fewer CH2− unit (δC 32.0; δH 1.83), the NMR data of 3 were similar to those of 2. The key 1 H−1H COSY correlations between H2-2 [δH 3.71 (2H, m)] and OH-2 [δH 4.84 (1H, br s)]/H2-3 [δH 2.87 (2H, m)] identified a spin system that consisted of C-2(OH)−C-3. The key HMBC correlations from H2-2 to C-4 and from H2-3 to C2/C-4/C-20 determined that a carbon was lost at the C-1 position. The planar structure of 3 was established by analyzing

Figure 1. Key 1H−1H COSY and HMBC correlations and planar structure of 1.

correlations (Figure 1a) from H-3 [δH 5.05 (1H, br dd, J = 8.9, 3.4 Hz)] to C-1/C-2/C-4/C-5/C-20, from H-20 [δH 8.06 (1H, br s)] to C-4/C-5/C-6, from H3-19 [δH 1.77 (3H, s)] to C-5/ C-9/C-10, from H-11 [δH 7.99 (1H, d, J = 8.1 Hz)] to C-8/C10/C-13, from H-12 [δH 7.93 (1H, d, J = 8.1 Hz)] to C-9/C14/C-17, from H-15a [δH 3.62 (1H, dt, J = 19.2, 5.7 Hz)]/H15b [δH 3.52 (1H, dt, J = 19.2, 5.7 Hz)] to C-14, and from H216 [δH 2.69 (2H, t, J = 5.7 Hz)] to C-17 revealed the partial structure of 1 (Figure 1a). On the basis of the NMR analysis, degrees of unsaturation, molecular formula, and chemical shifts of 1H and 13C, the planar structure of 1 was established as shown in Figure 1b, and the assignments of all proton and carbon resonances are provided in Table 1. The single-crystal X-ray crystallographic analysis (Figure 2) of 1 confirmed the above deduction. The value of the Flack parameter (0.09(19)) allowed the assignment of the absolute configuration of 1 as 3S, 10S. Therefore, the structure of 1 was B

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Table 1. NMR Spectroscopic Data for 1−5 1 position

δCa

1 2

58.1 40.8

δHa (J in Hz) 3.59, m a: 2.05, m

2 δ Ca 60.3 32.0

δHa (J in Hz) 3.51, m 1.83, m

3 δ Ca 60.4

δHa (J in Hz) 3.71, m

4 δ Ca 57.7 40.3

b: 1.87, m 3

61.5

4 5 6 7 8 9 10 11 12 13 14 15

131.2 140.5 144.6 173.2 126.9 158.4 69.3 127.1 126.8 137.0 156.4 27.8

16 17 19 20 OH-1 OH-2 OH-3 OH-10 a

36.1 205.6 32.6 146.7

5.05, br dd (8.9, 3.4)

7.99, d (8.1) 7.93, d (8.1)

a: 3.62, dt (19.2, 5.7) b: 3.52, dt (19.2, 5.7) 2.69, t (5.7) 1.77, s 8.06, br s

δHa (J in Hz) 3.60, m a: 2.02, m

5 δCa

δHa (J in Hz)

72.4 37.7

4.76, m a: 2.43, ddd (14.7, 7.6, 3.6) b: 2.16, ddd (14.7, 8.4, 1.8) 4.85, q (7.3)

b: 1.95, m 19.9 126.0 141.3 144.5 173.1 127.0 158.2 69.1 126.7 127.1 137.0 156.4 27.8

36.1 205.6 31.7 146.3

2.75, m

7.99, d (8.1) 7.93, d (8.1)

a: 3.62, dt (19.2, 5.7) b: 3.52, dt (19.2, 5.7) 2.69, t (5.7) 1.68, s 7.94, br s 4.56, t (5.0)

26.9 123.1 141.5 144.5 173.0 126.7 158.2 69.1 127.0 127.0, 137.1 156.4 27.8

36.1 205.6 32.0 146.9

2.87, m

7.99, d (8.1) 7.93, d (8.1)

a: 3.62, dt (19.2, 5.7) b: 3.53, dt (19.2, 5.7) 2.69, t (5.7) 1.68, s 7.95, br s

61.4 130.5 140.3 144.9 173.1 126.9 157.6 69.2 127.0 126.8 137.1 156.4 27.7

36.1 205.5 32.8 146.4

5.09, br dd (8.4, 4.3)

7.99, d (8.1) 7.92, d (8.1)

a: 3.62, dt (19.2, 5.7) b: 3.52, dt (19.2, 5.7) 2.68, t (5.7) 1.75, s 8.06, br s

58.5 125.3 145.0 144.9 172.9 130.9 156.7 43.1 125.9 125.8 136.2 157.5 28.1

7.82, d (8.0) 7.86, d (8.0)

a: 3.66, dt (19.0, 5.7) b: 3.54, dt (19.0, 5.7)

35.9 205.7 32.4 146.8

2.67, t (5.7) 1.47, s 8.07, br s 4.93, d (3.8)

4.84, br s 5.42, d (6.2) 6.29, s

6.35, s

The data were recorded in DMSO-d6 (1H NMR for 400 MHz, 13C NMR for 100 MHz).

Figure 2. X-ray structures of 1 and 6.

the 2D NMR data (HSQC, 1H−1H COSY, and HMBC) (Figures S18−S20, Supporting Information), which confirmed the above deduction. The assignments of all proton and carbon resonances are shown in Table 1. The ECD curve (Figure 3) of 3 was similar to that of 2, which suggested that 3 and 2 shared the same configuration at C-10. Therefore, the configuration of 3 was assigned as 10S, and the structure of 3 was elucidated as (S)-6-hydroxy-7-(2hydroxyethyl)-6-methyl-1H-cyclopenta[7,8]naphtho[2,3-b]furan-3,10(2H,6H)-dione and named nodulisporiviridin C. Compound 4, which was obtained as a yellowish, amorphous powder, was determined to have the identical molecular formula (C19H18O6) to that of 1. The 13C NMR spectrum disclosed 19 carbon signals, which corresponded to the

molecular formula. Combined with the DEPT experiment, these carbons can be categorized as two carbonyls (δC 205.5 and 173.1), 10 aromatic or olefinic carbons [including three sp2 methine carbons (δC 146.4, 127.0, and 126.8)], one oxygenated sp3 quaternary carbon (δC 69.2), one oxygenated sp3 methine carbon (δC 61.4), four sp3 methylene carbons [including an oxygenated carbon (δC 57.7)], and one methyl carbon (δC 32.8). The 1H NMR spectrum also showed that 4 possessed three olefinic or aromatic protons [δH 8.06 (1H, br s), 7.99 (1H, d, J = 8.1 Hz), and 7.92 (1H, d, J = 8.1 Hz)] and one methyl [δH 1.75 (3H, s)]. The planar structure of 4 was established according to the 1D NMR (1H NMR and 13C NMR) and 2D NMR (HSQC, 1H−1H COSY, and HMBC) (Figures S23−S25, Supporting Information), which was the C

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37.7, 35.9, and 28.1), and one methyl carbon (δC 32.4). The 1H NMR spectrum also revealed that 5 had three olefinic or aromatic protons [δH 8.07 (1H, br s), 7.86 (1H, d, J = 8.1 Hz), and 7.82 (1H, d, J = 8.1 Hz)] and one methyl [δH 1.47 (3H, s)]. The 1H NMR and 13C NMR data were similar to those of demethoxyviridin,14 which suggested the structural similarities for the two compounds. Compared with demethoxyviridin, 5 had an oxygenated methine carbon (δC 58.5) instead of a carbonyl (δC 190.2, C-3), which indicated that 5 was the carbonyl reduction derivative of demethoxyviridin at the C-3 position. The key 1H−1H COSY correlations identified a spin system that consisted of C-1(OH)−C-2−C-3(OH). The key HMBC correlations from H3-19 [δH 1.47 (3H, s)] to C-1/C-5/ C-9/C-10, from H-3 [δH 4.85 (1H, q, J = 7.3 Hz)] to C-4, and from H-20 [δH 8.07 (1H, br s)] to C-4/C-5/C-6 confirmed that the reduction occurred at the C-3 position. The planar structure of 5 was established based on the 2D NMR experiments (HSQC, 1H−1H COSY, and HSQC) (Figures S28−S31, Supporting Information), which confirmed the above deduction. The assignments of all proton and carbon resonances are shown in Table 1. In the ROESY experiment, the observed correlations between H3-19 and H-2b/OH-3 demonstrated that H-2b, H319, and OH-3 had identical orientations. The chemical shift of C-19 is obviously shifted to a lower field (δC 32.4) than that of demethoxyviridin (δC 25.3),14 which indicated that the relationship between H3-19 and H-1 in 5 was different from that of demethoxyviridin (1β-OH). In the selective 1D NOESY experiment (in DMSO-d6), an enhancement was observed for H3-19 in the irradiation of H-1, which determined the cis relationship between H3-19 and H-1. The small 3JH‑1−H‑2a (3.6 Hz) and 3JH‑1−H‑2b (1.8 Hz) confirmed the above deduction. On the basis of the above analysis, the relative configuration of 5 was established as shown in Figure 5.

Figure 3. Experimental ECD spectra of 1−4 in MeOH.

same as that of 1. The above NMR data analysis and further HPLC analysis (Figure 4) determined that 4 was an epimer of 1.

Figure 4. HPLC profiles of 1 and 4 under the same eluting systems (Phenomenex Gemini C18 column (4.6 × 250 mm, 5 μm), 18% MeCN−H2O (v/v), 1.0 mL/min).

In the ECD experiments, 1−3 (structural difference in C-1− C-2−C-3) displayed similar ECD curves, which indicated that the signs of the Cotton effects (∼240, ∼270, and ∼360 nm) of these metabolites are apparently controlled by the configuration of C-10. However, the ECD curve (Figure 3) of 4 was notably opposite that of 1, which suggested that the configuration of C10 in 4 was R. Considering that 4 was an epimer of 1, the absolute configuration of 4 was assigned as 3S, 10R. Therefore, the structure of 4 was established as (R)-7-((S)-1,3dihydroxypropyl)-6-hydroxy-6-methyl-1H-cyclopenta[7,8]naphtho[2,3-b]furan-3,10(2H,6H)-dione and named nodulisporiviridin D. Compound 5 was obtained as a white powder. The molecular formula was established as C19H16O5 (12 degrees of unsaturation) by the quasi-molecular ion at m/z 325.1076 [M + H]+ in the HRESIMS. The molecular weight of 5 had two more atomic mass units and one fewer degree of unsaturation than demethoxyviridin,14 which indicated that 5 may be a reduction derivative of demethoxyviridin. The 13C NMR spectrum showed 19 carbon signals, which corresponded to the molecular formula. Combined with the DEPT experiment, these carbons can be categorized as two carbonyls (δC 205.7 and 172.9), 10 aromatic or olefinic carbons [including three sp2 methine carbons (δC 146.8, 125.9, and 125.8)], one sp3 quaternary carbon (δC 43.1), two sp3 oxygenated methine carbons (δC 72.4 and 58.5), three sp3 methylene carbons (δC

Figure 5. Key ROESY correlations and selective 1D NOESY of 5.

The predicted ECD curves of (1S,3S,10R)-5 and (1R,3R,10S)-5 were calculated using a quantum chemical method at the [B3P86/6-311++G(2d,p)] level, and the predicted ECD curve of (1S,3S,10R)-5 was similar to the experimental one (Figure 6). Therefore, the absolute configuration of 5 was assigned as 1S, 3S, 10R, and the structure of 5 was elucidated as (1S,3S,11bR)-1,3-dihydroxy11b-methyl-1,2,3,7,8,11b-hexahydrocyclopenta[7,8]phenanthro[10,1-bc]furan-6,9-dione and named nodulisporiviridin E. Compound 6 was obtained as a colorless crystal plate. The quasi-molecular ion at m/z 343.1543 [M + H]+ by HRESIMS indicated that the molecular formula of 6 was C20H22O5 (10 degrees of unsaturation). The 13C NMR spectrum showed 20 D

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be assigned as 3S, 10R, 11S, 13S, 14R. Therefore, the structure of 6 was elucidated as (3S,6bR,9aS,11S,11bR)-3,11-dihydroxy9a,11b-dimethyl-1,2,3,7,8,10,11,11b-octahydrocyclopenta[7,8]phenanthro[10,1-bc]furan-6,9(6bH,9aH)-dione and named nodulisporiviridin F. Compound 7 was obtained as a yellowish powder, which was elucidated as C20H20O5 (11 degrees of unsaturation) by the HRESIMS (m/z 341.1384 [M + H]+), which was two atomic mass units fewer and one more degree of unsaturation than 6. The 13C NMR spectrum disclosed 20 carbon signals, which corresponded to the molecular formula. Combined with the DEPT experiment, these carbons can be categorized as three carbonyls (δC 217.4, 192.1, and 174.5), six aromatic or olefinic carbons [including one sp2 methine carbon (δC 148.5)], two sp3 quaternary carbons (δC 46.3 and 38.1), two sp3 methine carbons [including an oxygenated carbon (δC 66.6)], five sp3 methylene carbons, and two methyl carbons (δC 31.7 and 14.1). The 1H NMR spectrum also showed that 7 had one olefinic or aromatic proton [δH 8.65 (1H, s)] and two methyls [δH 0.92 (3H, s) and 1.72 (3H, s)]. The nonexchangeable proton resonances were associated with the directly attached carbon atoms in the HSQC experiment. Except for the absence of the oxygenated methine carbon (δC 61.0, C-3), the appearance of an additional carbonyl (δC 192.1), and the significantly different chemical shifts of C-1, C-2, and C-4, the NMR data were similar to those of 6, which indicated that 7 was a derivative of 6 with oxidation at C-3. The key 1H−1H COSY correlations identified a spin system that consisted of C-1−C-2. In HMBC, the key correlations from H3-19 [δH 1.72 (3H, s)] to C-1/C-5/ C-9/C-10, from H-1a [δH 2.71 (1H, ddd, J = 13.3, 5.5, 1.8 Hz)]/H-2a [δH 2.94 (1H, ddd, J = 18.6, 13.3, 5.3 Hz)] to C-3, and from H-20 [δH 8.65 (1H, s)] to C-4/C-5/C-6 confirmed that the oxidation occurred at the C-3 position. The planar structure of 7 was established using the 2D NMR experiments (HSQC, 1H−1H COSY, and HSQC) (Figures S40−S42, Supporting Information), which confirmed the above deduction. The assignments of all proton and carbon resonances are provided in Table 2. In the ROESY experiment, the observed correlations between H3-19 [δH 1.72 (3H, s)] and OH-11 [δH 5.62 (1H, d, J = 5.0 Hz)]/H3-18 [δH 0.92 (3H, s)] demonstrated that H318, OH-11, and H3-19 had identical orientations. The ROESY correlation between H3-18 and H-15b [δH 1.98 (1H, tt, J = 12.5, 8.5 Hz)] demonstrated that H3-18 and H-15b had identical orientations. In addition, the large 3JH−H (J = 12.5 Hz) of H-14 [δH 2.47 (1H, ddd, J = 12.5, 6.0, 2.0 Hz)] and H-15b indicated the trans relationship between H-14 and H-15b. On the basis of the above analysis, the relative configuration of 7 was established as shown in Figure 8a. The predicted ECD curves of (10R,11S,13S,14R)-7 and (10S,11R,13R,14S)-7 were calculated using a quantum chemical method at the [B3P86/6-311++G(2d,p)] level, and the predicted ECD curve of (10R,11S,13S,14R)-7 was similar to the experimental one (Figure 9). Thus, the absolute configuration of 7 was assigned as 10R, 11S, 13S, 14R, and the structure of 7 was elucidated as (9aS,11S,11bR)-11hydroxy-9a,11b-dimethyl-1,7,8,10,11,11b-hexahydrocyclopenta[7,8]phenanthro[10,1-bc]furan-3,6,9(2H,6bH,9aH)-trione and named nodulisporiviridin G. Compound 8 was isolated as a white powder with the molecular formula C20H22O5 as determined with HRESIMS, which indicated that 8 was an isomer of compound 6. Combined with the DEPT experiment, these carbons can be

Figure 6. Experimental ECD spectrum of 5 in MeOH and calculated ECD spectra of (1S,3S,10R)-5 and (1R,3R,10S)-5 (MeOH).

carbon signals, which corresponded to the molecular formula. Combined with the DEPT experiment, these carbons can be categorized as two carbonyls (δC 217.5 and 174.5), six aromatic or olefinic carbons [including one sp2 methine carbon (δC 145.4)], two sp3 quaternary carbons (δC 46.2 and 38.4), and three sp3 methine carbons [including two oxygenated carbons (δC 66.1 and 61.0)], five sp3 methylene carbons, and two methyl carbons (δC 31.7 and 14.1)]. The 1H NMR spectrum also showed that 6 had one olefinic or aromatic proton [δH 7.87 (1H, d, J = 1.0 Hz)] and two methyls [δH 1.67 (3H, s) and 0.92 (3H, s)]. All proton resonances were associated with the directly attached carbon atoms in the HSQC experiment. The analysis of the 1H−1H COSY experiment revealed three isolated spin systems (C-1−C-2−C-3, C-11−C-12, and C-

Figure 7. Key 1H−1H COSY and HMBC correlations and the planar structure of 6.

14−C-15−C-16), as shown in Figure 7a. Combined with the 1 H−1H COSY data, the HMBC correlations (Figure 7a) from H-3 [δH 4.65 1H, overlapped)] to C-4/C-5/C-20, from H-20 [δH 7.87 (1H, d, J = 1.0 Hz)] to C-4/C-5/C-6, from H3-19 [δH 1.67 (3H, s)] to C-1/C-5/C-9/C-10, from H3-18 [δH 0.92 (3H, s)] to C-12/C-13/C-14/C-17, from H-11 [δH 4.66 (1H, overlapped)] to C-8/C-9/C-13, from H-14 [δH 2.39 (1H, m)] to C-8, from H-15b [δH 1.92 (1H, m)] to C-14, and from H16a [δH 2.51 (1H, m)]/H-16b [δH 2.17 (1H, m)] to C-17 revealed the partial structure of 6 (Figure 7a). On the basis of the above NMR analysis, degrees of unsaturation, molecular formula, and chemical shifts of 1H and 13C, the planar structure of 6 was established (Figure 7b), and the assignments of all proton and carbon resonances are shown in Table 2. The single-crystal X-ray crystallographic analysis (Figure 2) of 6 confirmed the above deduction. The value of the Flack parameter (0.1(2)) enabled the absolute configuration of 6 to E

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Table 2. NMR Spectroscopic Data for 6−8 6 position

δCa

1

29.9

2

29.8

3 4 5 6 7 8 9 10 11

61.0 125.3 148.3 142.9 174.5 131.8 163.8 38.4 66.1

12

39.1

13 14 15

46.2 44.1 22.8

16

36.3

17 18 19 20 OH-3 OH-11 OH-12

217.5 14.1 31.7 145.4

δHa (J in Hz) a: 2.40, overlapped b: 1.66, overlapped a: 2.15, overlapped b: 1.94, m 4.65, overlapped

4.66, overlapped a: 1.78, m b: 1.65, overlapped 2.39, overlapped a: 2.90, br ddd (12.6, 8.0, 6.0) b: 1.92, m a: 2.51, m b: 2.17, overlapped 0.92, s 1.67, s 7.87, d (1.0)

7 δ Cb 30.4 36.2 192.1 121.6 148.7 143.8 174.2 132.1 163.4 38.1 66.6 39.3 46.3 43.9 22.7 36.3 217.4 14.3 28.5 148.5

δHb (J in Hz) a: 2.71, ddd (13.3, 5.5, 1.8) b: 2.07, td (13.3, 4.7) a: 2.94, ddd (18.6, 13.3, 5.3) b: 2.55, overlapped

4.70, m a: 1.78, m b: 1.78, m 2.47, ddd (12.5, 6.0, 2.0) a: 2.88, br ddd (12.5, 8.0, 6.0) b: 1.98, tt (12.5, 8.5) a: 2.54, overlapped b: 2.20, dt (19.3, 9.0) 0.92, s 1.72, s 8.65, s

8 δ Ca 31.2 30.1 60.6 125.3 146.5 143.5 173.8 131.0 163.5 37.9 34.5 67.4 50.7 42.7 22.3 36.4 218.1 7.4 29.3 145.5

δHa (J in Hz) a: 1.91, m b: 1.47, m a: 2.18, m b: 1.89, m 4.64, overlapped

a: 2.74, ddd (20.0, 7.8, 1.9) b: 2.24, ddd (20.0, 8.5, 2.7) 3.96, t (7.8)

2.63, m a: 2.88, br ddd (12.5, 8.2, 6.5) b: 1.95, tt (12.5, 8.8) a: 2.47, br dd (19.1, 8.4) b: 2.15, dt (19.1, 8.8) 0.82, 1.51, 7.89, 5.42,

s s d (0.8) br s

5.62, d (5.0) 4.66, overlapped

a

The data were recorded in DMSO-d6 (1H NMR for 400 MHz, 13C NMR for 100 MHz). bThe data were recorded in DMSO-d6 (1H NMR for 500 MHz, 13C NMR for 125 MHz).

Figure 8. Key ROESY correlations of 7 and 8.

categorized as two carbonyls (δC 218.1 and 173.8), six aromatic or olefinic carbons [including one sp2 methine carbon (δC 145.5)], two sp3 quaternary carbons (δC 50.7 and 37.9), three sp3 methine carbons [including two oxygenated carbons (δC 67.4 and 60.6)], five sp3 methylene carbons, and two methyl carbons (δC 29.3 and 7.4). The 1H NMR spectrum also showed that 8 had one olefinic or aromatic proton [δH 7.89 (1H, d, J = 0.8 Hz)] and two methyls [δH 0.82 (3H, s) and 1.51 (3H, s)]. The nonexchangeable proton resonances were associated with the directly attached carbon atoms in the HSQC experiment. The NMR spectra of 8 and 6 were similar, which indicated that 8 and 6 had identical skeletons. As observed in 6, the analysis of the 1H−1H COSY experiment of 8 revealed the presence of three isolated spin systems. However, the key HMBC correlations from H3-18 [δH 0.82 (3H, s)] to C-12/C-13/C14/C-17 suggested the presence of a hydroxy at C-12 for 8

Figure 9. Experimental ECD spectrum of 7 in MeOH and calculated ECD spectra of (10R,11S,13S,14R)-7 and (10S,11R,13R,14S)-7 (MeOH).

instead of at C-11 for 6. All 2D NMR experiments (HSQC, H−1H COSY, and HMBC) (Figures S46−S48, Supporting Information) supported the suggested structure of 8. Therefore, the planar structure of 8 was established, and the assignments of all proton and carbon resonances are provided in Table 2. In the ROESY experiment, the observed correlations between H3-18 and H3-19 [δH 1.51 (3H, s)]/H-11b [δH 2.24 1

F

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and ring C aromatization, and only a few have the ring A fission at C-2/C-3.18,20 Notably, nodulisporiviridins A−D (1−4), which were isolated from the strain of Nodulisporium sp. (No. 65-12-7-1) in the present study, are unique viridins with ring A fissured at C-1/C-10 with the 10R or 10S configuration. Inhibitory activities of compounds 1−8 against Aβ 42 aggregation were evaluated using the ThT assay with epigallocatechin gallate as a positive control (Table 3). EGCG, a polyphenol from green tea, prevents Aβ42 aggregation and is being tested in a phase 3 clinical trial of patients with early AD (NCT00951834).5,21

(1H, ddd, J = 20.0, 8.5, 2.7 Hz)] and between H3-19 and H-11b demonstrated that H3-18, H-11b, and H3-19 had identical orientations. The correlation between H3-18 and H-15b [δH 1.95 (1H, tt, J = 12.5, 8.8 Hz)] demonstrated that H3-18 and H-15b had identical orientations. The correlation between H12 [δH 3.96 (1H, t, J = 7.8 Hz)] and H-14 [δH 2.63 (1H, m)] demonstrated that H-12 and H-14 had identical orientations. In addition, the large 3JH‑14−H‑15b (J = 12.5 Hz) identified the trans relationship between H-14 and H-15b. H-3 changed its appearance to δH 4.66 (1H, t, J = 8.0 Hz) with the addition of D2O, and 3JH‑2a−H‑3 and 3JH‑2b−H‑3 were identical to those of 3-dihydrovirone (3β-OH, 3JH‑2a−H‑3 = 3JH‑2b−H‑3 = 8.0 Hz),16 which identified the cis relationship between OH-3 and C-19. Furthermore, the ROESY correlations between H-1b [δH 1.47 (1H, m)] and H-11a [δH 2.74 (1H, ddd, J = 20.0, 7.8, 1.9 Hz)]/ H-3[δH 4.64 (1H, overlapped)] demonstrate that H-11a, H-1b, and H-3 had identical orientations, which confirmed the cis relationship between OH-3 and C-19. On the basis of the above analysis, the relative configuration of 8 was established as shown in Figure 8b. The predicted ECD curves of (3S,10R,12R,13R,14S)-8 and (3R,10S,12S,13S,14R)-8 were calculated using a quantum chemical method at the [B3P86/6-311++G(2d,p)] level, and the predicted ECD curve of (3S,10R,12R,13R,14S)-8 was similar to the experimental one (Figure 10). Therefore, the

Table 3. Aβ42 Aggregation Inhibitory Activities of 1−8 ThT assay compound EGCG 1 2 3 4 5 6 7 8

relative inhibitory activity (%) (100 μM)

IC50a (μM)

± ± ± ± ± ± ± ± ±

0.5 95.9 28.0 34.3 71.9 10.1

95.1 63.5 89.5 62.8 80.5 99.0 54.6 98.8 70.4

9.3 8.2 2.5 11.3 5.7 0.1 6.2 0.3 4.9

1.2 43.5

When the relative inhibitory activities of compounds were ≥60.0%, the IC50 values were evaluated. a

The short-term memory enhancement activities of 1−8 were evaluated using the human Aβ42 transgenic AD fly model with memantine as the positive control. The activities of these compounds were indicated with the performance index (PI) (see Figure 11). The positive control memantine is a

Figure 10. Experimental ECD spectrum of 8 in MeOH and calculated ECD spectra of (3S,10R,12R,13S,14S)-8 and (3R,10S,12S,13R,14R)-8 (MeOH).

Figure 11. Performance index (PI) of AD flies fed with compounds 1−8. The treated groups were AD flies treated with mematine or test compounds (100 μM), and the control groups (2U*H29.3 represents the normal files, and P35*H29.3 represents the AD files) were treated with a corresponding volume of DMSO. (a) Statistical analysis results of 1 and 2. (b) Statistical analysis results of 3−5. (c) Statistical analysis results of 6−8. Each value is expressed as mean ± SEM, n = 8; *p < 0.001, significantly different from the normal group; #p < 0.001, significantly different from the AD group; t-test.

absolute configuration of 8 was assigned as 3S, 10R, 12R, 13R, 14S, and the structure of 8 was elucidated as (3S,6bS,9aR,10R,11bR)-3,10-dihydroxy-9a,11b-dimethyl1,2,3,7,8,10,11,11b-octahydrocyclopenta[7,8]phenanthro[10,1bc]furan-6,9(6bH,9aH)-dione and named nodulisporiviridin H. In most cases, proton−proton couplings over more than three bonds, particularly couplings across five bonds, are usually too small to be easily detected (95%, GL Biochem, Shanghai) was dissolved in 10 μL of dimethyl sulfoxide (DMSO, purity >99%, ACROS, USA) and 544 μL of phosphate-buffered saline (PBS, 0.01 mol/L, pH 6.6) to a concentration of 40 μM. Thioflavin T (purity >95%, Sigma) was dissolved in PBS to a concentration of 80 μM. Then 12.5 μL of the different concentrations of samples, 25 μL of the Aβ42 stock solution, and 12.5 μL of ThT were added to 96-well black plates (Costar, USA) with shaking at 90 rpm for 15 min. The mixture was incubated at 37 °C for 16 h, and the fluorescence was measured using the Acumen Explorer (Acumen X3, TTP Labtech Ltd., UK). The dose-dependent inhibition of compounds against Aβ42 aggregates was monitored in three individual experiments. EGCG (purity >95%, Sigma) was used as a positive control. Biological activity was determined as relative inhibitory activity (Vi) for each sample according to the formula Vi = [(F0 − Fi)/F0] × 100, where Fi and F0 are the fluorescence value of Aβ42 aggregation with and without the test sample, respectively. For active inhibitor (relative inhibitory activity ≥60%), the inhibitory constant (IC50) was determined by testing in six concentrations in three independent experiments. The statistical analysis was performed using Graph Pad 5.03 for Windows. For the determination of IC50 concentrations, the mean percent inhibition dose−response curves were fitted to the dose−response inhibition [log (inhibitor) vs response-variable slope (four parameters)]. The dose−response equation is Y = Bottom + (Top − Bottom)/[1 + 10(log IC50 − X) × Hillslope], where X is the compound concentration, Y is the Vi, top and bottom are the plateaus in the units of the y-axis, and Hillslope describes the steepness of the family of curves. Fly Stock.26,27 w1118 (isoCJ1) is an isogenic line used as a control in all of the experiments. We named this stock “2U” for convenience. Expression of Aβ42 (UAS-Aβ42; referred to as H29.3) was driven by a ubiquitous neuronal-expressing Gal4 line, elav-GAL4c155 (P35). A behavioral assay was made from the first generation of the cross between P35 × H29.3. Fly Culture.26 All flies were reared at 24 °C, 42% RH (relative humidity). On day 1, newly hatched 2U*H29.3 male flies and AD male flies were selected and were put into vials (each vial contains about 120 flies). Those flies were placed at 28 °C, 42% RH during the drug feeding process. The flies were transferred to new vials after 4 h of drug feeding from day 2 to day 8. All flies were placed at 28 °C, 42% RH until 1 h before the Pavlovian olfactory learning assay. Drug Feeding Schedule.26 Drugs (compounds 1−8 and memantine) were prepared on the first day of eclosion, and the drug feeding was implemented on day 2. The original concentration of compounds was 10 mM, and the final concentration of compounds was 100 μM. For each performance index, 2 vials of flies were fed with 50 μL of the resulting solution for 7 days (e.g., from day 2 to day 8). Due to the fact that some flies died naturally during these processes, on day 9, about 100 flies were left in each vial for further Pavlovian olfactory learning assay, which was also done on day 9. Pavlovian Olfactory Learning.28−30 Briefly, during one training session, a group of about 100 flies was exposed sequentially to two odors (either octanol or methylcyclohexanol) for 60 s with a 45 s rest interval after each odor presentation. During exposure to the first odor, flies were simultaneously subjected to foot shock (1.5 s pulses with 3.5 s intervals, 60 V). To measure immediate memory (also referred to as learning), flies were transferred immediately after training to the choice point of a T-maze and given a choice between the two odors for 2 min, after which they were trapped in their respective arms,

Table 1; ESIMS (positive) m/z 325 [M + H]+, 649 [2 M + H]+; ESIMS (negative) m/z 323 [M − H]−, 647 [2 M − H]−; HRESIMS (positive) m/z 325.1076 [M + H]+ (calcd for C19H17O5, 325.1076). Nodulisporiviridin F (6): colorless crystal plate (MeOH); mp 286− 290 °C; [α]29D −16.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 203 (3.90), 220 (3.75), 261 (3.91), 315 (3.81) nm; ECD (c 2.9 × 10−4 M, MeOH) λmax (Δε) 209 (+1.78), 225 (−5.60), 246 (+2.61), 271 (−8.97), 307 (+4.78), 357 (−0.81); IR (KBr) νmax 3509, 3361, 2958, 2934, 2863, 1728, 1649, 1614, 1444, 1397, 1338, 1074, 1057 cm−1; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) see Table 2; ESIMS (positive) m/z 365 [M + Na]+, 707 [2 M + Na]+; HRESIMS (positive) m/z 343.1543 [M + H]+ (calcd for C20H23O5, 343.1545). Nodulisporiviridin G (7): yellowish powder; mp 176−179 °C; [α]29D −31.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 205 (4.18), 231 (3.96), 261 (3.91), 302 (3.75) nm; ECD (c 2.9 × 10−4 M, MeOH) λmax (Δε) 224 (−4.66), 267 (−6.28), 305 (+3.02); IR (KBr) νmax 3390, 3094, 2931, 2869, 1742, 1694, 1619, 1517, 1473, 1146, 1106 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) see Table 2; ESIMS (positive) m/z 363 [M + Na]+, 703 [2 M + Na]+; ESIMS (negative) m/z 339 [M − H]−, 375 [M + Cl]−; HRESIMS (positive) m/z 341.1384 [M + H]+ (calcd for C20H21O5, 341.1389). Nodulisporiviridin H (8): white powder; mp 254−256 °C; [α]29D −44.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 204 (3.97), 220 (3.81), 261 (3.97), 310 (3.86) nm; ECD (c 2.9 × 10−4 M, MeOH) λmax (Δ ε) 209 (+3.79), 226 (−4.30), 246 (+0.89), 268 (−3.04), 307 (+3.59), 348 (−0.95); IR (KBr) νmax 3421, 2922, 2851, 1729, 1658, 1618, 1458, 1420, 1380, 1040 cm−1; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) see Table 2; ESIMS (positive) m/z 343 [M + H]+; ESIMS (negative) m/z 341 [M − H]−; HRESIMS (positive) m/z 343.1550 [M + H]+ (calcd for C20H23O5, 343.1545). X-ray Crystallographic Analysis of 1. Upon crystallization from MeOH using the vapor diffusion method, a plate of 1 was obtained. Data were collected using a Sapphire CCD with a graphitemonochromated Cu Kα radiation, λ = 1.541 84 Å at 173.0(3) K. Crystal data: C19H18O6 ·H2O, M = 360.35, monoclinic, space group P21; unit cell dimensions were determined to be a = 7.2425(3) Å, b = 8.2512(3) Å, c = 13.6931(5) Å, α = 90.00°, β = 96.661(3)°, γ = 90.00°, V = 812.76(5) Å3, Z = 2, Dx = 1.472 g/cm3, F(000) = 380, μ(Cu Kα) = 0.947 mm−1; 12 633 reflections were collected until θmax = 62.80°, in which independent unique 2359 reflections were observed [F2 > 4σ(F2)]. The structure was solved by direct methods using the SHELXS-97 program and refined by the SHELXL-97 program and full-matrix least-squares calculations.24 In the structure refinements, non-hydrogen atoms were placed on the geometrically ideal positions by the “ride on” method. Hydrogen atoms bonded to oxygen were located by the structure factors with isotropic temperature factors. The final refinement gave R = 0.0311, Rw = 0.0759, S = 1.094, Flack = 0.09(19), and Hooft y = 0.05(9). Crystallographic data for nodulisporiviridin A (1) have been deposited in the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 1033899. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033, or e-mail: [email protected]. uk). X-ray Crystallographic Analysis of 6. Upon crystallization from MeOH using the vapor diffusion method, a plate of 6 was obtained. Data were collected using a Sapphire CCD with a graphitemonochromated Cu Kα radiation, λ = 1.541 78 Å at 173.01(11) K. Crystal data: C20H22O5, M = 342.38, monoclinic, space group P21; unit cell dimensions were determined to be a = 8.4907(3) Å, b = 8.5157(2) Å, c = 12.1520(4) Å, α = 90.00°, β = 109.005(4)°, γ = 90.00°, V = 830.75(4) Å3, Z = 2, Dx = 1.369 g/cm3, F(000) = 364, μ(Cu Kα) = 0.802 mm−1; 9077 reflections were collected until θmax = 60.82°, in which 2431 independent unique reflections were observed [F2 > 4σ(F2)]. The structure was solved by direct methods using the SHELXS-97 program and refined by the SHELXL-97 program and full-matrix least-squares calculations.24 In the structure refinements, non-hydrogen atoms were placed on the geometrically ideal positions I

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anesthetized, and counted. A PI was calculated from the distribution of flies in the T-maze. PI = 0 represents a 50:50 distribution, which means the flies cannot remember one odor with foot shock, whereas PI = 100 represents 100% of flies avoided the shock-paired odor by running into the other T-maze arm. Note: The learning assay was carried out in a dark room at 24 °C, 70% RH. Flies were moved into the dark room 1 h before the assay, so that the flies could get familiar with the environment. Statistical Analysis. Data were analyzed (Table S10, Supporting Information) and graphs (Figure 11) were also plotted with the program Graph Pad 5.03.

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ASSOCIATED CONTENT

* Supporting Information S

NMR assignments of 1−8, quantum chemical ECD calculation of 5, 7, and 8, the result of short-term memory assay of 1−8 on the human Aβ42 transgenic AD flies, and the 1D and 2D NMR spectra of 1−8. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ np500912t.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-20-85221559. E-mail: [email protected]. Author Contributions ∥

Q. Zhao and G.-D. Chen have contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from the Ministry of Science and Technology of China (2012ZX09301002-003001006), the National Natural Science Foundation of China (81422054, 81373306, 81202441, 81172945), the Guangdong Natural Science Funds for Distinguished Young Scholar (S2013050014287), the Science and Technology Program of Guangzhou (2013J4501037), and Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (H.G., 2014). We also thank Joekai Biotech. Co., Ltd. for the short-term memory assay, American Journal Experts Inc. for the linguistic corrections, and the highperformance computing platform of Jinan University for the ECD calculations.

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REFERENCES

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