Bialternacins AâF, Aromatic Polyketide Dimers from an Endophytic

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Bialternacins A−F, Aromatic Polyketide Dimers from an Endophytic Alternaria sp. Cheng Long Yang,†,∥ Hui Min Wu,†,∥ Cheng Li Liu,† Xuan Zhang,† Zhi Kai Guo,§ Yu Chen,# Fang Liu,# Yong Liang,# Rui Hua Jiao,† Ren Xiang Tan,†,‡ and Hui Ming Ge*,†

J. Nat. Prod. Downloaded from pubs.acs.org by WEBSTER UNIV on 02/22/19. For personal use only.

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State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, Nanjing 210023, People’s Republic of China ‡ State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China § Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, People’s Republic of China # State Key Laboratory of Coordination Chemistry, Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, People’s Republic of China S Supporting Information *

ABSTRACT: Six novel aromatic polyketide dimers, bialternacins A−F (1−6), were isolated from a plant endophytic Alternaria sp. The structures of compounds 1−6 were elucidated on the basis of extensive spectroscopic analysis, single-crystal X-ray diffraction, and electronic circular dichroism analysis. Compounds 1, 2, 5, and 6 were characterized as four pairs of racemic mixtures. Compound (+)-5 was demonstrated to show acetylcholinesterase inhibitory activity with an IC50 value of 15.5 μM. A putative biosynthetic pathway for these compounds was proposed.

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biological evaluation, and the possible biosynthetic pathway for these compounds.

ungi in the genus Alternaria (Dematiaceae) are globally distributed plant pathogens, which cause severe crop spoilage and extensive plant diseases.1−4 In addition, they have been shown to be lethal to poultry and have been implicated in diseases of many mammals. The mycotoxins from Alternaria have been investigated extensively, leading to the identification of an array of secondary metabolites including dibenzopyrone, anthraquinones, tetramic acids, and fusicoccins,4−8 which were demonstrated to show diverse bioactivities including cytotoxicity to plant and mammalian cells and antiviral and enzyme inhibitory activities. In recent years, symbiotic fungi have drawn substantial attention due to their potential to produce chemically diverse biologically active secondary metabolites. In the course of our continuing efforts to explore the metabolic potential of symbiotic fungi,9−13 we isolated a fungus, NF2128, belonging to the genus Altanaria, from the stem of a traditional Chinese medicinal plant, Maianthemum bifolium, collected in Emei Mountain of Sichuan Province of China. After cultivation in a production medium at 28 °C for 2 weeks, the fermentation broth was extracted with ethyl acetate and analyzed by HPLCUV/MS. This strain produced two major metabolites (1 and 2) with molecular weights of 559 and 558, respectively, both of which have not been reported from an Altanaria sp. To identify these unknown secondary metabolites, the fermentation was scaled up and extracted, leading to the isolation of six novel aromatic polyketide dimers, bialternacins A−F (1−6). We report here details of the isolation, structural characterization, © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Bialternacin A (1) was isolated as yellow crystals. Its molecular formula of C30H25NO10 was established by high-resolution ESIMS and its 13C NMR, corresponding to 19 degrees of unsaturation. The IR spectrum of 1 indicated the presence of carbonyl (1728 and 1668 cm−1) and hydroxy (3499 cm−1) functionalities. Analysis of its 1H, 13C, and HSQC spectral data of 1 (Table 1) revealed the presence of two exchangeable protons (δH 11.30 and 7.60) and seven olefinic/aromatic protons (δH 6.71, 7.25, 6.11, 6.47, 7.16, 7.15, and 6.64), suggesting the presence of aromatic rings. In addition, one pair of methylene protons (δH 2.67 and 2.87), two singlet methoxyl protons (δH 3.77 and 3.92), and two singlet methyl protons (δH 2.00 and 1.68) were observed. Two 1,2,3,5-tetrasubstituted phenyl rings and a 1,2,4,5tetrasubstituted phenyl ring were readily determined by analysis of the 1H−1H COSY and HMBC data (Figure 1). A cyclohexene ring in 1 was established by HMBC correlations from H-3′ (δH 2.67) to C-5′ (δC 154.8) and C-7′ (δC 141.2), from H-1′ (δH 1.68) to C-3′ (δC 45.4) and C-7′ (δC 141.2), from H-6′ (δH 7.16) to C-2′ (δC 81.3) and C-5′ (δC 154.8), and from 4′-OH Received: August 21, 2018

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DOI: 10.1021/acs.jnatprod.8b00705 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

optical activity. Compound 2 was then separated on a chiral column to yield enantiomers (+)-2 and (−)-2 with optical rotation values of [α]25D +67 (c 0.02, MeOH) and −80 (c 0.02, MeOH), respectively. Their absolute configurations were readily determined by comparison of the ECD with the experimental data (Figure 4). Structural analysis of 1 and 2 indicated they could be dimerized from two biphenyl units. The distinct unusual dimerization encouraged us to further isolate more precursors/congeners for understanding their putative biosynthetic pathway. Our attention has mainly focused on the compounds with similar molecular weight to those of 1 and 2 in the remaining fractions. The subsequent fractionation and purification guided by LC-MS led to the characterization of three minor metabolites, 3−5. The molecular formula of 3 was determined to be C30H26O12 based on its HRESIMS data. The 13C NMR spectrum displayed 15 carbon signals, suggesting a symmetric nature of 3. The chemical shifts of carbon signals were assignable to a methyl group, a methoxyl group, a carboxylic carbon, and 12 sp2-hybrized carbons resembling those found in the right half substructure (marked in black) of 2. Detailed HMBC (Figure S2) analysis confirmed the presence of a biphenyl ring connected via the C-7−C-8 position. Importantly, the HMBC correlations from OH-4 (δH 8.92) to C-4 (δC 142.4), from OH-5 (δH 7.55) to C-5 (δC 141.5), from OH-12 (δH 12.50) to C-12 (δC 162.8), from OCH3 (δH 3.59) to C-10 (δC 162.7), and from H-1 (δH 1.68) to C-2 (δC 145.3), C-3 (δC 115.5), and C-7 (δC 125.1) confirmed that the biphenyl unit in 3 has the same substituted pattern as the right half substructure in 2. Finally, similar to 2, the two symmetric biphenyl units were connected through C-6−C-6′, which is the only remaining unassigned carbon. It is notable that 3 can slowly convert to three major compounds, 2, 4, and 5, as well as a minor one, 6. The molecular formula of 4 was determined to be C30H24O11. Compound 4 is also a symmetric structure, as indicated by only 15 carbon signals being observed in its 13C NMR spectrum. The NMR data of 4 are almost identical to those in the right half structure of 2, indicating a similar substructure and substitution pattern. The detailed HMBC analysis (Figure S2) finally established the structure of 4, as a dehydration product of 3 (Scheme 1). Compound 5 has the molecular formula C30H24O12 as deduced by HRESIMS. The 1H and 13C NMR data resembled those in 2. A biphenyl substructure (marked in black) is revealed by NMR comparison with those in 3 and 2D NMR analysis (Table 1 and Figure S2). Moreover, the HMBC correlations of H-1′ (δH 1.44) with C-2′ (δC 78.8), C-3′ (δC 115.2), and C-7′ (δC 143.9) and of H-3′ (δH 5.90) with C-4′ (δC 146.6), C-5′ (δC 181.1), C-6′ (δC 135.0), and C-7′ (δC 143.9) indicated the presence of a 4-methylcyclohexa-2,5-dien-1-one group. Further HMBC analysis established the planar structure of 5. 5 is also a racemic mixture, whose absolute configurations were solved by comparison of calculated ECD with experimental data (Figure 5). The structure of 6 is directly determined by analysis of its single-crystal X-ray data (Scheme 1). The crystal of 6 has the space group P21/C, indicating 6 is also a racemic mixture. However, the NMR data of 6 could not be collected due to insufficient material. Based on the structural features of 1−6, a plausible biosynthetic pathway was postulated. The formation of biphenyl polyketide monomer was initiated by successive decarboxylative Claisen condensation of one acetyl CoA and six malonyl CoA units catalyzed by fungal polyketide synthase, followed by

(δH 7.60) to C-3′ (δC 45.4) and C-5′ (δC 154.8). In addition, the HMBC correlations from H-6′ (δH 7.16) to C-8′ (δC 136.3), from H-9′ (δH 7.15) to C-7′ (δC 141.2), and from H-9 (δH 6.11) to C-7 (δC 129.0) indicated that the above four rings are connected via C-7′−C-8′ and C-7−C-8, respectively (Figure 1). The construction of the complete structure of 1 solely by NMR analysis, however, proved to be challenging due to the lack of key correlations to connect the above deduced substructures. A single crystal was successfully obtained upon slow evaporation of the solvent at room temperature for 2 weeks. The gross structure of 1 was constructed as an aromatic polyketide dimer with an unprecedented 6/6/6/6/6/6-hexacyclic scaffold. Notably, the specific optical rotation value of 1 was close to zero, suggesting a racemic mixture. Subsequent chiral HPLC separation of 1 was performed to afford a pair of enantiomers with 1:1 ratio (Figure S1) with almost opposite specific optical rotation values. To assign the absolute configuration of (+)-1 and (−)-1, electronic circular dichroism (ECD) spectra were generated on the basis of quantum-chemical calculations. The ECD spectrum for (2′S,4′R)-1 agrees well with that measured for (−)-1, whereas, (2′R,4′S)-1 matches well with (+)-1 (Figure 2). Thus, the absolute configurations of both enantiomers were determined.14 Compound 2 was obtained as pale yellow crystals and possessed a molecular formula of C30H22O11 on the basis of HRESIMS. Similar to the structure of 1, compound 2 also possessed a biphenyl substructure (marked in black in Figure 1) connected through a single C-7−C-8 bond based on the analysis of its 1H−1H COSY and HMBC spectra. The HMBC correlations from H-1′ (δH 1.50) to C-2′ (δC 157.6), C-3′ (δC 127.8), C-4′ (δC 175.8), and C-7′ (δC 82.5) and from H-3′ (δH 6.28) to C-5′ (δC 149.5) and C-7′ (δC 82.5) revealed the presence of a cyclohexa-2,5-dien-1-one moiety, which was connected to a 1,2,3,5-tetrasubstituted phenyl ring via C-7′−C8′ as deduced by HMBC correlation of H-9′ (δH 6.25) with C-7′ (δC 82.5) (Figure 3). The remaining two unsaturated degrees were proposed to be consumed by the formation of two additional rings, but this was difficult to assign by NMR analysis. This puzzle was finally solved by analysis of its single-crystal Xray data (Figure 3). 2 is a pair of enantiomers based on its lack of B



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Table 1. NMR Data of Bialternacins A−E (1−5) 1 (DMSO-d6) no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 10-OMe 12-OH 4-OH 5-OH 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 10′-OMe 12′-OH 4′-OH 5′-OH

δH (J in Hz) 2.00, s 6.71, s

7.25, s

6.11, brs 6.47, brs

3.77, s

1.68, s 2.67, d (12.8) 2.87, d (12.8)

7.16, s

7.15, d (2.0) 6.64, s

3.92, s 11.3, s 7.60 brs

δC, type

2 (acetone-d6) δH (J in Hz)

18.9, CH3 142.5, C 116.1, CH 132.8, C 144.0, C 127.5, CH 129.0, C 143.5, C 108.1, CH 162.2, C 100.2, CH 162.9, C 108.1, C 171.4, C 55.4, CH3

1.75, s

26.8, CH3 81.3, C 45.4, CH2

1.50, s

88.2, C 154.8, C 123.8, CH 141.2, C 136.3, C 103.5, CH 166.0, C 103.1, CH 163.3, C 99.8, C 166.9, C 56.1, CH3

6.91, s

6.17, d (2.4) 6.39, d (2.4)

3.90, s

6.28, d (1.2)

6.25, d (1.2) 6.36, d (1.8)

3.71, s

δC, type 20.2, CH3 144.3, C 117.0, CH 143.9, C 142.5, C 125.3, C 128.7, C 133.0, C 111.4, CH 165.2, C 102.6, CH 165.9, C 105.2, C 172.6, C 55.7, CH3

17.4, CH3 157.6, C 127.8, CH 175.8, C 149.5, C 127.3, C 82.5, C 150.9, C 100.7, CH 167.9, C 103.8, CH 158.1, C 106.2, C 169.0, C 56.4, CH3

3 (DMSO-d6) δH (J in Hz) 1.68, s 6.41, s

6.38, d (2.4) 6.10, d (2.4)

3.59, s 12.50, s 8.92, s 7.55, s 1.68, s 6.41, s

6.38, d (2.4) 6.10, d (2.4)

3.59, s 12.50, s 7.60, s 7.55, s

δC, type

4 (acetone-d6) δH (J in Hz)


1.73, s

20.2, CH3 145.3, C 115.5, CH

1.73, s

142.4, C 141.5, C 124.7, C 125.1, C 132.8, C 108.9, CH 162.7, C 100.7, CH 164.8, C 106.1, C 173.3, C 55.2, CH3

6.83, s

5.83, d (2.4) 6.20, d (2.4)

3.71, s

6.83, s

5.83, d (2.4) 6.20, d (2.4)

3.71, s

δC, type

5 (DMSO-d6) δH (J in Hz)


1.74, s

20.6, CH3 144.6, C 115.7, CH

1.44, s

145.7, C 141.6, C 124.6, C 130.1, C 130.5, C 112.2, CH 164.2, C 100.5, CH 165.7, C 106.3, C 173.2, C 55.4, CH3

6.61, s

5.57, d (2.0) 6.21, d (2.0)

3.64, s

5.90, s

6.45, d (2.2) 6.44, d (2.2)

3.61, s

δC, type 19.3, CH3 132.9, C 116.3, CH 142.9, C 142.4, C 125.8, C 120.0, C 131.5, C 108.0, CH 162.6, C 100.6, CH 164.7, C 104.7, C 172.0, C 55.4, CH3


be dimerized via a C−C bond to form compound 3 through intermolecular oxidative phenol coupling catalyzed most likely by a P450 monooxygenase or laccase.16−19 Dehydration of 3 gave compound 4. Oxidation of catechol in 3 afforded intermediate V, which can further undergo a regioselective intramolecular Michael additions to afford either γ-lactone intermediate VI or δ-lactone intermediate VII. Dehydration of VI afforded 2. Ketone−enol tautomerization gave 5, which can be oxidized to 6. Acetylcholinesterase (AChE) is a promising target for treatment of Alzheimer’s disease, as inhibition of the activity of AChE leads to an increase in the level of acetylcholine, a neurotransmitter playing a key role in memory.20 All isolated compounds were thus subjected to in vitro AChE inhibitory evaluations. 4 and (+)-5 showed inhibitory activity with IC50 values of 68.3 and 15.5 μM, respectively, whereas the other compounds were all inactive.

Figure 1. Key HMBC correlations and crystal structure of 1.

regioselective cycloaddition and postmodification that gave monomer I. The catechol groups are prone to oxidize to an ortho-benzoquinone moiety.15 A subsequent intramolecular Michael addition gave intermediate III, which could further coupled with I via a morpholine ring to yield 1. Meanwhile, I can

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were collected on a Bruker Avance 400 or 600 NMR spectrometer. Crystal data were C



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Figure 2. Comparison of experimental and calculated ECD spectra of (+)-1 and (−)-1. flasks containing 200 mL of ME medium (containing 20 g malt, boil 30 min, take filtrate; 1 g peptone in 1 L water) followed by cultivation at 28 °C with 140 rpm agitation for 2 days. The seed cultures were then inoculated to 100 × 1 L flasks each containing 400 mL of ME medium and fermented for 14 days at 28 °C with 140 rpm agitation. Extraction and Isolation. The obtained crude extract was fractionated using silica gel column chromatography (CC) and eluted with petroleum ether−ethyl acetate (100:0, 100:2, 100:4, 100:8, 100:10, 100:20, 100:50, 1:1, 0:100, v/v), CH2Cl2−MeOH (100:0, 100:4, 100:10, 100:20, 100:50, 0:100, v/v) to afford 16 fractions (A−P) after TLC monitoring. Fractions D and E were further fractionated using ODC CC and eluted with MeOH−H2O (30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 100:0, v/v) to afford seven fractions respectively (d-1, d-2, d-3, d-4, d-5, d-6, d-7, e-1, e-2, e-3, e-4, e-5, e-6, e-7). Fraction d-2, d-4, and e-4 were further separated by Sephadex LH-20 with MeOH to afford six fractions, respectively (d-2-1, d-2-2, d-2-3, d-2-4, d2-5, d-2-6, d-4-1, d-4-2, d-4-3, d-4-4, d-4-5, d-4-6, e-4-1, e-4-2, e-4-3, e4-4, e-4-5, e-4-6). Subsequently, fraction d-4-5 was further purified by semipreparative HPLC [acetonitrile−H2O−formic acid (45:55:0.1), 2 mL/min, wavelength at 254 nm] to yield 1; fraction e-4-2 was further purified by semipreparative HPLC [acetonitrile−H2O−formic acid (43:57:0.1), 2 mL/min, wavelength at 254 nm] to yield 2, 3, and 4; fraction d-2-4 was further purified by semipreparative HPLC [acetonitrile−H2O−formic acid (38:62:0.1), 2 mL/min, wavelength at 254 nm] to yield 5. During purification of 5, a single crystal (6) was obtained, which allowed us to directly measure its single-crystal X-ray data. Bialternacin A (1): yellow crystal; mp 208−210 °C; [α]25D −80 for (−)-1 (c 0.01, MeOH) and [α]25D +60 for (+)-1 (c 0.01, MeOH); UV/

Figure 3. Key HMBC correlations and crystal structure of 2. acquired on a Bruker APEX-II CCD single-crystal diffractometer using Cu Kα radiation at low temperature. All structures were analyzed by SHELXS-97 software and refined by means of full-matrix least-squares. High-resolution LC-MS data were obtained on an Agilent 6520 TOF mass spectrometer. UV spectra were recorded on a Thermo Nanodrop2000c spectrometer. Optical rotation values were measured at a Rudolph Research Analytic Autopol IV automatic polarimeter. Semipreparative HPLC was performed on an Agilent 1260 LC machine equipped with an Eclipse XDB-C18 column (5 μM, 250 × 9.4 mm; Agilent Technologies Inc. USA). Strain Isolation and Cultivation. Strain NF2128 was isolated from the leaves of Maianthemum bifolium collected from Emei Mountain of Sichuan Province and belongs to the genus Alternaria based on its ITS sequence (accession no. MK138621). The purified strain was cultivated in a PDA medium plate (containing 200 g potatoes, boil 30 min, take filtrate; 20 g sucrose; 20 g agar in 1 L water) at 28 °C for 7 days. Then the fresh spores were inoculated in 10 × 1 L

Figure 4. Comparison of experimental and calculated ECD spectra of (+)-2 and (−)-2. D



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Scheme 1. Proposed Biosynthetic Pathway for 1−6

Figure 5. Comparison of experimental and calculated ECD spectra of (+)-5 and (−)-5. vis (MeOH) λmax (log ε) 218 (4.79), 389 (4.89), 259 (4.41) nm; HRESIMS m/z 560.1550 [M + H]+ (calcd for C30H25NO10H+: 560.1551); 1H and 13C NMR data, see Table 1. Bialternacin B (2): light yellow crystal; mp 265−267 °C; [α]25D −75 for (−)-2 (c 0.02, MeOH) and [α]25D +67 for (+)-2 (c 0.02, MeOH); UV/vis (MeOH) λmax (log ε) 228 (4.78), 300 (4.77), 335 (4.35) nm; HRESIMS m/z 581.1053 [M + Na]+ (calcd for C30H22O11Na+: 581.1054); 1H and 13C NMR data, see Table 1. Bialternacin C (3): yellow, amorphous powder; UV/vis (MeOH) λmax (log ε) 216 (4.63), 256 (4.48), 396 (4.42) nm; HRESIMS m/z 601.1311 [M + Na]+ (calcd for C30H22O12Na+: 601.1316); 1H and 13C NMR data, see Table 1. Bialternacin D (4): white, amorphous powder; UV/vis (MeOH) λmax (log ε) 228 (4.78), 300 (4.70), 335 (4.35) nm; HRESIMS m/z 583.1206 [M + Na]+ (calcd for C30H24O11Na+: 583.1211); 1H and 13C NMR data, see Table 1. Bialternacin E (5): yellow amorphous powder; [α]25D −70 for (−)-5 (c 0.20, MeOH) and [α]25D +77 for (+)-5 (c 0.20, MeOH); UV/vis (MeOH) λmax (log ε) 218 (4.76), 248 (4.69), 303 (4.31) nm; HRESIMS m/z 599.1156 [M + Na]+ (calcd for C30H24O12Na+: 599.1160); 1H and 13C NMR data, see Table 1. Bialternacin F (6): light yellow crystal; mp 218−220 °C; HRESIMS m/z 597.1002 [M + Na]+ (calcd for C30H22O12Na+: 597.1004). Crystal Data for Compounds 1, 2, and 6. Crystal data of 1: C30H25NO10, MW = 559.51, monoclinic, a = 17.7022(6) Å, b = 9.1991(3) Å, c = 19.6833(6) Å, β = 115.412(2)°, V = 2895.18(17) Å3, T = 173.01 K, space group P21/n, Z = 4, μ(Cu Kα) = 0.819, reflections

collected 51 014, wR2 (all data) = 0.2158. The crystal data of 1 have been deposited in the CCDC under accession no. 1844149. Crystal data of 2: C30H22O11, MW = 558.47, triclinic, a = 9.5566(5) Å, b = 11.1116(6) Å, c = 13.2868(7) Å, α = 78.022(3)°, β = 86.311(3)°, γ = 79.060(3)°, V = 1354.65(13) Å3, T = 153 K, space group P1̅, Z = 2, μ(Cu Kα) = 0.894, reflections collected 7293, wR2 (all data) = 0.0730. The crystal data of 2 have been deposited in the CCDC under accession no. 1844150. Crystal data of 6: C30H22O12, MW = 574.47, monoclinic, a = 14.7453(3) Å, b = 14.0183(3) Å, c = 15.5000(3) Å, β = 115.3140(10)°, V = 2896.27(10) Å3, T = 304.11 K, space group P21/c, Z = 4, μ(Cu Kα) = 0.559, reflections collected 9826, wR2 (all data) = 0.1358. The crystal data of 6 have been deposited in the CCDC under accession no. 1844148. Chiral Separation of 1, 2, and 5. Compound 1 was separated by a Shimadzu LC-20A on a Daicel Chiralpak IB chiral column (Daicel Chemical Industries, Ltd., Japan). The mobile phase contained hexane/ EtOH/trifluoroacetic acid (40:60:0.1) with a flow rate of 1.0 mL/min (Figure S1). Compounds 2 and 5 were separated by a Shimadzu LC20A on a Daicel Chiralpak IE chiral column. The mobile phase consisted of hexane−EtOH−trifluoroacetic acid (70:30:0.1 and 75:25:0.1) with a flow rate of 1.0 mL/min (Figure S1). Computation Methods for ECD of Compounds 1, 2, and 5. For compounds 1, 2, and 5, we assigned the absolute configurations by comparing their measured ECD spectra with the density functional theory (DFT)-computed ones. This technique has been successfully applied to the assignment of absolute configuration of many natural E



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products. ECD calculations were performed with the Gaussian 09 software package. The geometric optimization was carried out at the B3LYP/TZVP level of theory. Frequency analyses were performed at the same level of theory to confirm that all the stationary points are minima (with no imaginary frequency). The solvation effect (in methanol) was evaluated using the PCM model during geometric optimization. ECD spectra were computed via time-dependent DFT (NStates = 30) at the same level of theory. The calculated ECD spectra of compounds 1, 2, and 5 were corrected with blue shifts of 20, 24, and 29 nm, respectively. The values of blue shifts were obtained from the differences of main peaks between the computed UV spectra and those from experiments. Acetylcholinesterase Inhibitory Activity. The AChE inhibitory activity was determined by a classical spectrophotometric method.21 Briefly, the enzymatic reaction was conducted in a 200 μL volume reaction system consisting of 0.35 U/mL AChE (Sigma-Aldrich), 3.33 mM 5,5′-dithiobis(2-nitrobenzoic acid) (Sigma-Aldrich, Shanghai, China), and 5.30 mM acetylthiocholine iodide (Sigma-Aldrich) in 96-well microplates. DMSO solutions of the tested compounds were added to the assay solution and incubated for 5 min, followed by the measurement of absorbance at 412 nm with a plate reader. Five different concentrations and three repetitions per concentration for each compound were used to measure the inhibition of AChE activity with huperzine A, an acetylcholinesterase inhibitor isolated from Huperzia serrata,22 as positive control (IC50 4.3 μM). All test and control assays were corrected by blanks for nonenzymic hydrolysis. CCDC 1844149, 1844150, and 1844148 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by e-mailing data_ [email protected], or by contacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033.

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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.8b00705. Experimental procedures; NMR spectroscopic data (PDF) X-ray crystallization data for compound 1 (CIF) X-ray crystallization data for compound 2 (CIF) X-ray crystallization data for compound 6 (CIF)

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail (H. M. Ge): [email protected]. ORCID

Zhi Kai Guo: 0000-0002-9608-0847 Fang Liu: 0000-0002-0046-8434 Yong Liang: 0000-0001-5026-6710 Ren Xiang Tan: 0000-0001-6532-6261 Hui Ming Ge: 0000-0002-0468-808X Author Contributions ∥

C. L. Yang and H. M. Wu contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Natural Science Foundation of China (21572100, 81522042, 81773591, 81500059, 81673333, 21672101, and 21661140001) and Fundamental Research Funds for the Central Universities (020814380092). F


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