α-Glucosidase Inhibitors from the Marine-Derived Fungus Aspergillus

Note pubs.acs.org/jnp

α‑Glucosidase Inhibitors from the Marine-Derived Fungus Aspergillus flavipes HN4-13 Cong Wang,†,§ Lei Guo,†,‡,§ Jiejie Hao,† Liping Wang,⊥ and Weiming Zhu*,† †

Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Jiangsu Marine Resources Development Research Institute, School of Marine Life and Fisheries, Huaihai Institute of Technology, Lianyungang 222004, People’s Republic of China ⊥ Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550002, People’s Republic of China S Supporting Information *

ABSTRACT: Three new butenolide derivatives, flavipesolides A−C (1−3), along with 13 known compounds (4−13, aspulvinone Q, monochlorosulochrin, and dihydrogeodin), were isolated from the marine-derived Aspergillus f lavipes HN4-13 from a Lianyungang coastal sediment sample. The structures were elucidated by spectroscopic evidence. Compounds 4−6 and 9 were noncompetitive α-glucosidase inhibitors with Ki/IC50 values of 0.43/ 34, 2.1/37, 0.79/19, and 2.8/90 μM, respectively. Compounds 1−3, 8, 10, and 13 are mixed α-glucosidase inhibitors with Ki/IC50 values of (2.5, 19)/44, (3.4, 14)/57, (9.2, 4.7)/95, (6.3, 5.5)/55, (1.4, 0.60)/9.9, and (2.5, 7.2)/33 μM, respectively (IC50 101 μM for acarbose and 79 μM for 1-deoxynojirimycin). respectively. The 1H NMR signals of δH 7.40 (d, J = 8.4 Hz, 2H) and 6.80 (d, J = 8.4 Hz, 2H) showed the presence of a psubstituted phenyl moiety. Three aromatic signals at δH 6.89 (s, 1H), 6.86 (d, J = 8.3 Hz, 1H), and 6.68 (d, J = 8.3 Hz, 1H) indicated the presence of a 1,3,4-trisubstituted benzene ring. These assignments were further supported by the COSY correlations (Figure 1) of H-2′ and H-6′ (δH 7.40)/H-3′ and

H

aving complex genetic backgrounds and high yields of secondary metabolites, marine-derived fungi are becoming a rich source of new marine natural products (MNPs),1 and marine-derived Aspergillus are the most productive fungi for new MNPs.2 Moreover, the secondary metabolites of Aspergillus fungi exhibit antitumor, insecticidal, antimicrobial, and antioxidant activities.2 In a continuing chemical investigation on new metabolites from marine Aspergillus fungi,3 Aspergillus f lavipes HN4-13 was isolated and identified from a Lianyungang coastal sea sediment sample,4 Jiangsu Province of China. A chemical study on its secondary metabolites led to the identification of three new butenolide derivatives, named flavipesolides A−C (1−3), as well as 13 known compounds, 5-[(3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-6-yl)methyl]-3hydroxy-4-(4-hydroxyphenyl)-2(5H)furanone (4),5 aspernolide A (5),6 emodin (6),7 questin (7),7 geodin hydrate (8),7 methyl dichloroasterrate (9),8 monomethylosoic acid (10),9 asterric acid (11),7 methyl 3-chloroasterric acid (12),8 epicoccolide B (13),10 aspulvinone Q,11 monochlorosulochrin,7 and dihydrogeodin12 (Tables S2−S5, Supporting Information). Compounds 1−6, 8−10, and 13 exhibited stronger inhibition of α-glucosidase and lower cytotoxicity to Caco-2 cell than the positive controls 1-deoxynojirimycin and acarbose. Compound 1 was obtained as a light brown solid. Its molecular formula was determined as C25H26O7 from the HRESIMS peak at m/z 439.1751 [M + H]+, indicating 13 degrees of unsaturation. The UV absorptions at 210, 230, and 310 nm were similar to those of 4, indicating a butenolide skeleton. 6 The IR absorption at 1758 and 3386 cm −1 corresponded to γ-lactone carbonyl and hydroxy groups, © 2016 American Chemical Society and American Society of Pharmacognosy

Figure 1. Key COSY and HMBC correlations of compound 1.

H-5′ (δH 6.80), and H-5″ (δH 6.68)/H-6″ (δH 6.86). According to the DEPT and HMQC spectra, the 13C NMR spectrum consisted of 25 carbon signals including three methyls, four methylenes, seven sp2-methines, and 11 nonprotonated carbons. The HMBC showed long-range 1H−13C correlations (Figures 1 and S6−S8) from H-2″ (δH 6.89) and H-6″ (δH 6.86) to C-6 (δC 29.4), from H-2″ to C-6″ (δC 126.9) and oxygenated C-4″ (δC 152.8), and from H-5″ (δ 6.68) to C-1″ (δC 128.6)/C-3″ (δC 121.9), indicating the 1,3,4-trisubstituted benzene ring linked as a 3,4-disubstituted benzyl group. The Received: August 20, 2016 Published: November 14, 2016 2977

DOI: 10.1021/acs.jnatprod.6b00766 J. Nat. Prod. 2016, 79, 2977−2981

Journal of Natural Products

Note

rotation with 1 indicated the same configuration (Figure S33). Thus, compound 2, named flavipesolide B, was elucidated as (S)-methyl 4-((2,2-dimethylchroman-6-yl)methyl)-2-hydroxy3-(4-hydroxyphenyl)-5-oxo-2,5-dihydrofuran-2-carboxylate. This compound might be an artifact of 3 probably arising from the use of MeOH in the large-scale isolation process. Compound 3 also showed similar UV, IR, and NMR data and COSY and HMBC coupling patterns, implying a homologue of 1 and 2. The insolubility in CDCl3 and deshielded shifts of C-1−C-4, when compared with the NMR data of 1 (Figures S9−11) and 2 (Figures S20−22) in DMSOd6, indicated that 3 might be a carboxylic acid or salt. The negative ion HRESIMS peak of 3 at m/z 409.1293 [M − H]− (calcd 409.1293 for the anion C23H21O7−) along with the positive ion HRESIMS peak at m/z 411.1433 [M + H]+ (calcd 411.1438 for the cation C23H23O7+) supported 3 as a carboxylic acid. The formation of 3 from the basic hydrolysis of 1 further consolidated the structure. Thus, flavipesolide C (3) could be identified as (S)-4-((2,2-dimethylchroman-6-yl)methyl)-2-hydroxy-3-(4-hydroxyphenyl)-5-oxo-2,5-dihydrofuran-2-carboxylic acid. Because no cytotoxicity against MCF-7, HepG2, Jurkat, A549, and K562 tumor cells was observed with an inhibition less than 34% at 10 μM (Table S1), α-glucosidase inhibition of compounds 1−10, 12, 13, aspulvinone Q, monochlorosulochrin, and dihydrogeodin was assayed using p-nitrophenyl-αglucopyranoside (pNPG) as the substrate.14 The cytotoxicity against the Caco-2 cell line was assayed by the MTT method.15 The results showed that compounds 1−6, 8−10, and 13 exhibited stronger inhibition of α-glucosidase and lower cytotoxicity to the Caco-2 cell line than 1-deoxynojirimycin (IC50 79 ± 2 μM) and acarbose (IC50 101 ± 4 μM) with IC50 values of 44 ± 2, 57 ± 1, 95 ± 3, 34 ± 1, 37 ± 1, 19 ± 1, 55 ± 2, 90 ± 3, 9.9 ± 0.3, and 33 ± 1 μM, respectively, while compounds 7, 12, aspulvinone Q, monochlorosulochrin, and dihydrogeodin were not active against α-glucosidase (IC50 120−1300 μM). The median cytotoxic concentrations (CC50) were 519 and 278 μM for 1 and 2 and >800 μM for compounds 3−6, 8−10, and 13, respectively. Thus, the selectivity indexes (SI = CC50/IC50) of compounds 1−6, 8− 10, and 13 were 12, 4.8, >8.5, >24, >22, >43, >15, >8.9, >81, and >24, respectively. On the basis of these data, the enzyme kinetics were further studied.16 Results indicated that compounds 4−6 and 9 were noncompetitive α-glucosidase inhibitors with Ki values of 0.43, 2.1, 0.79, and 2.8 μM, respectively (Figure S34). However, compounds 1−3, 8, 10, and 13 showed mixed behavior with Ki values of 2.5/19, 3.4/14, 9.2/4.7, 6.3/5.5, 1.4/0.60, and 2.5/7.2 μM, respectively (Figure S35). Mixed inhibition indicates both competitive and noncompetitive inhibitions, and the two Ki values in turn represented the competitive and noncompetitive inhibitions. Compared with 3, esterified compounds 1 and 2 showed stronger α-glucosidase inhibition. The comparison of 7 with 6 indicated that O-methylation at 1-OH caused decreased αglucosidase inhibition by the anthraquinones, and the benzophenone derivatives showed less inhibition of αglucosidase than the diphenyl ether compounds. α-Glucosidase inhibitors, such as acarbose and miglitol, have good therapeutic effects in clinical application for the treatment of diabetes.16 However, most of them have side effects such as nausea and diarrhea, so safer and more effective inhibitors are urgently needed. This study offers different α-glucosidase inhibitors with

HMBC correlations from H-1‴ to C-3‴ (δC 74.5)/C-2″−C-4″, from H-2‴ to C-3″/C-3‴−C-5‴, and from H-4‴ and H-5‴ (δH 1.31) to C-2‴ (δC 32.9)/C-3‴/C-5‴ (δC 27.0), along with the COSY correlations of H-1‴ (δH 2.70)/H-2‴ (δH 1.77) (Figure S5), further linked this benzyl group as a (2,2-dimethyl-3,4dihydro-2H)-pyrano[2,3-c]benzyl moiety. The HMBC correlations from H2-6 (δH 3.81, 3.77) to three nonprotonated carbons, C-1 (δC 171.9), C-2 (δC 127.8), and C-3 (δC 154.3), connected the benzyl group to C-2 of the γ-butenolide nucleus. The key HMBC correlations from H-2′ and H-6′ to C-3 (δC 154.3) linked the p-substituted phenyl to C-3 of the γbutenolide nucleus. The chemical shift of C-4 (δC 100.4) indicates a ketal or hemiketal carbon that connected with an ethoxycarbonyl on the basis of a COSY connection of methylene protons (δH 4.24) with methyl protons (δH 1.19) and the key HMBC correlation of the methylene protons (δH 4.24) to a carbonyl carbon (δC 168.3). The remaining two hydrogen and two oxygen atoms implied that C-4 must be a hemiketal carbon and the p-substituted phenyl is a phydroxyphenyl. Thus, the constitution of compound 1 was elucidated as ethyl 4-((2, 2-dimethylchroman-6-yl)methyl)-2hydroxy-3-(4-hydroxyphenyl)-5-oxo-2,5-dihydrofuran-2-carboxylate. The absolute configuration was determined by comparison of the measured ECD spectrum with the calculated ECD spectrum using the TD-DFT method (Supporting Information).13 The results indicated that the measured ECD spectrum of 1 matched well with the calculated ECD spectrum for S-1 and is opposite that of R-1 (Figure 2). The Cotton effect arising

Figure 2. Calculated ECD curves of S-1 and R-1 and the measured ECD curve of 1.

from the π−π* transition is more applicable than that of the n−π* transition in elucidating the absolute configuration of α,β-unsaturated γ-lactones. The obvious positive ECD Cotton effect at 207 nm (+2.6) clearly indicated the S-configuration of the butenolide nucleus. Compound 1 was named flavipesolide A and was identified as (S)-ethyl 4-((2,2-dimethylchroman-6yl)methyl)-2-hydroxy-3-(4-hydroxyphenyl)-5-oxo-2,5-dihydrofuran-2-carboxylate. The molecular formula of compound 2 was deduced as C24H24O7 from the HRESIMS peak at m/z 425.1595 [M + H]+, one CH2 unit less than 1. The similar NMR (Table 1), COSY (Figure S16), and HMBC patterns (Figures S17−S19) indicated a homologue of 1. Careful NMR comparison of 2 with 1 further indicated a methylene group less than 1, and a methoxy signal (δOCH3 54.7/3.80) in 2, indicated a 5-methyl ester in 2. This deduction was further supported by an HMBC correlation of the methoxy protons (δH 3.80) with the carbonyl carbon (δC 168.9). The similar ECD spectrum and specific 2978

DOI: 10.1021/acs.jnatprod.6b00766 J. Nat. Prod. 2016, 79, 2977−2981

Journal of Natural Products

Note

Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for Compounds 1−3a 1 position 1 2 3 4 5 6 1′ 2′/6′ 3′/5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴/5‴

δC, type 171.9, 127.8, 154.3, 100.4, 168.3, 29.4, 121.3, 130.4, 116.1, 157.9, 128.6, 129.2, 121.9, 152.8, 117.5, 126.9, 22.6, 32.9, 74.5, 27.0,

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

2 δH (J in Hz)

3.81, d (15.5); 3.77, d (15.5) 7.40, d (8.4) 6.80, d (8.4)

6.89, s

6.68, 6.86, 2.70, 1.77,

d (8.3) d (8.3) t (6.8) t (6.8)

1.31, s

δC, type 171.8, 127.8, 154.0, 100.4, 168.9, 29.5, 121.9, 130.4, 116.1, 157.9, 128.7, 129.2, 121.3, 152.8, 117.6, 127.1, 22.6, 32.9, 74.5, 27.0,

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

3 δH (J in Hz)

3.81, d (15.5); 3.76, d (15.5) 7.38, d (8.4) 6.82, d (8.4)

6.88, s

6.69, 6.87, 2.70, 1.76,

d (8.1) d (8.1) t (6.6) t (6.6)

1.31, s

δC, type 172.9, 128.7, 157.6, 103.3, 167.4, 28.7, 120.7, 130.1, 115.2, 158.7, 124.1, 128.9, 121.9, 152.0, 116.6, 126.6, 21.9, 32.1, 73.8, 26.6,

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

δH (J in Hz)

3.67, d (15.7); 3.63, d (15.7) 7.35, d (8.3) 6.76, d (8.3)

6.94, s

6.60, 6.90, 2.66, 1.72,

d (8.4) d (8.4) t (6.6) t (6.6)

1.24, s

The δ values were recorded in CDCl3 for 1 and 2 and in DMSO-d6 for 3. The δC/H of 5-OCH2CH3 for 1 and 5-OCH3 for 2 were 64.3 (CH2)/4.24 (q, J = 7.1 Hz, 2H) and 13.9 (CH3)/1.19 (t, J = 7.1 Hz, 3H), and 54.7 (CH3)/3.80 (s, 3H), respectively. The δH of 4′-OH for 3 is 9.92 (s, 1H). a

Chart 1

■

strong inhibitions and low toxicity to Caco-2 cells for further study. Phenyl- and benzyl-disubstituted γ-butenolides are a special kind of 7,8-dimeric phenylpropanoid. This kind of natural products (NPs) is mainly produced by fungi especially from Aspergillus sp.,17−21 but is seldom found in plants22 and animals.23 These NPs have shown anti-TMV,17 DPPH scavenging,18 α-glucosidase inhibition,19 antimalaria,20 and anti-H1N1 virus activities.21 According to the substituted positions of the phenyl and benzyl groups, their structures can be divided into three types, that is, 2,3-, 3,4-, and 2,4disubstituted γ-butenolides. The 2,4- and 3,4-disubstituted types are common γ-butenolides from nature. The 2,3disubstituted type is a rare γ-butenolide both from nature and synthesis, with only two papers reporting a natural one (butyrolactone VIII)21 and 10 synthetic ones.24 Compounds 1−3 represent the second report of naturally occurring 2,3disubstituted γ-butenolides. Compounds 1−3 are also the first examples of 2,3-disubstituted γ-butenolide α-glucosidase inhibitors.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were acquired on a JASCO P-1020 digital polarimeter, UV spectra were recorded on a Beckman DU 640 spectrophotometer, and ECD data were collected on a JASCO J-715 spectropolarimeter. IR spectra were taken on a Nicolet Nexus 470 spectrophotometer as KBr disks. The NMR spectra were obtained using a JEOL JNM-ECP 600 spectrometer or a Bruker Avance 500 spectrometer and were referenced to the residual solvent signals (CHCl3 δH/C 7.26/77.16, DMSO δH/C 2.50/39.52). HRESIMS spectra were collected on a QTOF Ultima Global GAA076 LC mass spectrometer. Semipreparative HPLC was performed using a Cosmosil cholester column (Cosmosilpack, 10 × 250 mm, 5 μm, 4 mL/min) and an ODS column (YMCpack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min). Vacuum-liquid chromatography (VLC) utilized silica gel H (Qingdao Marine Chemical Factory). TLC and column chromatography were carried out by plates precoated with silica gel GF254 (10−40 μm, Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Biosciences), respectively. The natural seawater used was from the Lianyungang sea area of the Yellow Sea. Fungal Material and Fermentation. The fungus Aspergillus f lavipes HN4-13 was isolated from a sediment sample collected in the Lianyungang coastal area, Jiangsu Province of China. The strain was 2979

DOI: 10.1021/acs.jnatprod.6b00766 J. Nat. Prod. 2016, 79, 2977−2981

Journal of Natural Products

Note

from expression of the recombinant gene in the yeast Saccharomyces cerevisiae was used. Each compound was dissolved in sodium phosphate buffer (PBS, pH 6.8) at three concentrations. A volume of 10 μL of the compound, 20 μL of PBS, and 30 μL of 2.5 mM pNPG solution were mixed in a 96-well microplate and incubated at 37 °C for 15 min. A volume of 10 μL of α-glucosidase diluted to 0.2 U/mL by 0.01 M PBS was then added to each well. After incubating at 37 °C for 15 min, the absorbance at 405 nm was recorded by a Spectra Max 190 micro plate reader (Molecular Devices Inc.). The blank was prepared by adding PBS instead of the α-glucosidase, and the positive controls were acarbose and 1-deoxynojirimycin. Blank readings (no enzyme) were subtracted from each well, and the results were compared to the control. The inhibition (%) was calculated as [1 − (ODdrug/ODblank)] × 100%. The half-maximal inhibitory concentration (IC50) was calculated as the compound concentration that is required for 50% inhibition, and the IC50 values of the acarbose and 1-deoxynojirimycin were 101 and 79 μM, respectively. Kinetics of α-Glucosidase Assay. According to refs 14 and 16, the inhibitory modes on α-glucosidase of compounds 1−6, 8−10, and 13 were measured with increasing concentrations of pNPG (0, 0.75, 1, 1.25, 2.5, 5, 8, or 10 mM) as a substrate in the absence and presence of the corresponding compounds as described in the text. Optimal amounts of tested compounds were determined on the basis of the αglucosidase inhibition assay. The mode of inhibition was resolved by Lineweaver−Burk plot analysis of the data that were calculated by Michaelis−Menten kinetics.

prepared on potato dextrose agar medium and identified by one of the authors (L.G.) according to its morphological properties and ITS sequence analysis (GenBank access no. JX287370). Spores were transferred to 100 × 1000 mL Erlenmeyer flasks consisting of 80 g of rice and 120 mL of natural seawater (pH 7.0). Extraction and Isolation. The solid culture was extracted three times with EtOAc to give EtOAc solutions. The EtOAc solutions were combined and evaporated in vacuo to yield 60.1 g of an EtOAc extract. The EtOAc extract was separated into 12 fractions on a silica gel VLC column by step gradient elution with CH2Cl2−petroleum ether (50− 100%) and then with MeOH−CH2Cl2 (0−50%). Fraction 5 (2.0 g) was purified over Sephadex LH-20 into four subfractions, eluting with MeOH−CH2Cl2 (1:1). Subfraction 5-2 (500 mg) was further purified by semipreparative HPLC (65% MeOH−H2O, 1.5‰ trifluoroacetic acid (TFA), 4.0 mL/min) over a Cosmosil-pack cholester column to yield 1 (22 mg, tR 15.3 min), 2 (17 mg, tR 12.6 min) and 3 (16 mg, tR 5.0 min). Subfraction 5−3 (478 mg) was further purified by semipreparative HPLC (65% MeOH−H2O, 1.5‰ TFA, 4.0 mL/ min) over a Cosmosil-pack cholester column to yield 4 (17 mg, tR 13.5 min) and 5 (10 mg, tR 15.8 min). Subfraction 5−4 (672 mg) was further purified by semipreparative HPLC (45% MeCN−H2O, 1.5‰ TFA, 4.0 mL/min) over a Cosmosil-pack cholester column to yield 9 (80 mg, tR 16.5 min). Fraction 9 (231 mg) was purified by HPLC over a Cosmosil-pack cholester column (45% MeCN−H2O, v/v, 1.5‰ TFA, 4.0 mL/min) to yield aspulvinone Q (3 mg, tR 6.5 min) and 10 (8 mg, tR 5.8 min). Fraction 6 (5.0 g) was purified over Sephadex LH20 into three subfractions, eluting with MeOH−CH2Cl2 (v/v 1:1). Subfraction 6-2 (2.2 g) was further purified by HPLC over a Cosmosilpack cholester column (60% MeOH−H2O, v/v, 1.5‰ TFA, 4.0 mL/ min) to yield 6 (tR 6.7 min, 7.0 mg), 7 (tR 8.3 min, 20 mg), 8 (tR 5.7 min, 3.2 mg), monochlorosulochrin (tR 7.4 min, 13 mg), and dihydrogeodin (tR 5.2 min, 3.7 mg). Subfraction 6-3 (366 mg) was purified over a Cosmosil-pack cholester column (60% MeCN−H2O, v/v, 1.5‰ TFA, 4.0 mL/min) by HPLC to yield 11 (tR 9.4 min, 3 mg) and 12 (tR 14.0 min, 3.6 mg). Fraction 10 (350 mg) was purified over a Cosmosil-pack cholester column (65% MeOH−H2O, v/v, 1.5‰ TFA, 4.0 mL/min) by HPLC to yield 13 (11 mg, tR 14.7 min). Flavipesolide A (1): light brown solid; [α]25D −28 (c 0.1, CH2Cl2); UV (MeOH) λmax (log ε) 210 (2.60), 230 (2.43), and 310 (2.31) nm; ECD (0.0023 M, MeOH) λmax (Δε) 207 (+2.6) nm; IR (KBr) νmax 3386, 2970, 2933, 1608, 1758, 1516, 1497, 1431, 1350, 1260, 1122, 1035, 1000, 943, 840, 808 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 439.1751 [M + H]+ (calcd for C25H27O7, 439.1750). Flavipesolide A (2): light brown solid; [α]25D −34 (c 0.1, CH2Cl2); UV (MeOH) λmax (log ε) 202 (2.61), 228 (2.39), and 308 (3.98) nm; ECD (0.0024 M, MeOH) λmax (Δε) 207 (+2.7) nm; IR (KBr) νmax 3386, 2970, 2935, 1759, 1607, 1518, 1499, 1436, 1385, 1259, 1201, 1120, 1066, 1035, 943, 840, 755 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 425.1583 [M + H]+ (calcd for C24H25O7, 425.1595). Flavipesolide A (3): light brown solid; [α]25D −22 (c 0.1, CH2Cl2); UV (MeOH) λmax (log ε) 204 (3.11), 226 (1.21) and 306 (0.81) nm; ECD (0.0024 M, MeOH) λmax (Δε) 207 (+2.9) nm; IR (KBr) νmax 3421, 1684, 1515, 1500, 1430, 1350, 1250, 1120, 1035, 1000, 947, 845, 810 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 411.1433 [M + H]+ (calcd for C23H23O7, 411.1438), m/z 409.1293 [M − H]− (calcd for C23H21O7, 409.1293). Chemical Transformation of 1 to 3. Compound 1 (1.2 mg) reacted with KOH (1.0 mg) and in MeOH (2 mL) at the room temperature (rt) for 3.5 h under a nitrogen atmosphere until 1 was consumed. The reaction mixture was cooled to rt and was neutralized to pH 5 by 0.33 N HCl. The reaction mixture was directly purified by HPLC (65% MeOH−H2O, 4.0 mL/min) over a Cosmosil-pack cholester column to afford 3 (0.8 mg, tR 9.7 min). The product was identified by ESIMS (m/z 411.2 [M + H]+, 433.2 [M + Na]+), specific rotation ([α]25D −18 (c 0.08, CH2Cl2), and the same 1H NMR spectrum (Figure S31) and retention time in the co-HPLC profiles as natural 3 (Figure S32). α-Glucosidase Inhibition Assay. Glucosidase inhibition was assayed as described previously.14,16 Human α-glucosidase obtained

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00766. Bioassay protocols used, the NMR spectra of compounds 1−3, and the NMR data of compounds 4−13, aspulvinone Q, monochlorosulochrin, and dihydrogeodin; measured ECD curves of 1−3; Lineweaver−Burk plots of α-glucosidase inhibition of 1−6, 8−10, and 13 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel (W. Zhu): +86-532-82031268. Fax: +86-532-82031268. E-mail: [email protected]. ORCID

Weiming Zhu: 0000-0002-7591-3264 Author Contributions §

C. Wang and L. Guo contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by grants from the NSFC (Nos. 41376148 and 81561148012), from the NSFCGuangdong Joint Fund for Key Projects (No. U1501221), from the NSFC-Shandong Joint Fund for Marine Science Research Centers (No. U1406402), and from the Special Fund for Marine Scientific Research in the Public Interest of China (No. 201405038).

■

REFERENCES

(1) Pietra, F. Nat. Prod. Rep. 1997, 14, 453−464. (2) Zhao, C.; Zhu, T.; Zhu, W. Youji Huaxue 2013, 33, 1195−1234. (3) Zheng, J.; Xu, Z.; Wang, Y.; Hong, K.; Liu, P.; Zhu, W. J. Nat. Prod. 2010, 73, 1133−1137. 2980

DOI: 10.1021/acs.jnatprod.6b00766 J. Nat. Prod. 2016, 79, 2977−2981

Journal of Natural Products

Note

(4) Guo, L.; Zhu, W. C.; Liu, W. W.; Ding, G. W.; Zhang, Y. Q.; Zhang, S. L.; Qiu, Y.; Xie, Y. Microbiol. China 2013, 40, 951−958. (5) Nitta, K.; Fujita, N.; Yoshimura, T.; Arai, K.; Yamamoto, Y. Chem. Pharm. Bull. 1983, 31, 1528−1533. (6) Kiriyama, N.; Nitta, K.; Sakaguchi, Y.; Taguchi, Y.; Yamamoto, Y. Chem. Pharm. Bull. 1977, 25, 2593−2601. (7) Liu, D.; Yan, L.; Ma, L.; Huang, Y.; Pan, X.; Liu, W.; Lv, Z. Arch. Pharmacal Res. 2015, 38, 1038−1043. (8) Hargreaves, J.; Park, J.; Ghisalberti, E. L.; Sivasithamparam, K.; Skelton, B. W.; White, A. H. J. Nat. Prod. 2002, 65, 7−10. (9) Nateri, S.; Nishikawa, I. Chem. Pharm. Bull. 1962, 10, 117−124. (10) Li, W.; Yang, X.; Yang, Y.; Duang, R.; Chen, G.; Li, X.; Li, Q.; Qin, S.; Li, S.; Zhao, L.; Ding, Z. Nat. Prod. Res. 2016, 30, 1−4. (11) Zhang, L. H.; Feng, B. M.; Zhao, Y. Q.; Sun, Y.; Liu, B.; Chen, G.; Bai, J.; Hua, H. M.; Wang, H. F.; Pei, Y. H. Bioorg. Med. Chem. Lett. 2016, 26, 346−350. (12) Sato, S.; Okusa, N.; Ogawa, A.; Ikenoue, T.; Seki, T.; Tsuji, T. J. Antibiot. 2005, 58, 583−589. (13) Stephens, P. J.; Pan, J. J.; Krohn, K. J. J. Org. Chem. 2007, 72, 7641−7649. (14) Kawamura-Konishi, Y.; Watanabe, N.; Saito, M.; Nakajima, N.; Sakaki, T.; Katayama, T.; Enomoto, T. J. Agric. Food Chem. 2012, 60, 5565−5570. (15) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (16) Chen, Z.; Hao, J.; Wang, L.; Wang, Y.; Kong, F.; Zhu, W. Sci. Rep. 2016, 6, 20004. (17) Zhou, M.; Lou, J.; Li, Y. K.; Wang, Y. D.; Zhou, K.; Ji, B. K.; Dong, W.; Gao, X. M.; Du, G.; Hu, Q. F. J. Braz. Chem. Soc. 2015, 26, 545−549. (18) Zhang, P.; Li, X. M.; Wang, J. N.; Li, X.; Wang, B. G. Phytochem. Lett. 2015, 11, 85−88. (19) Zhang, L. H.; Feng, B. M.; Zhao, Y. Q.; Sun, Y.; Liu, B.; Liu, F.; Chen, G.; Bai, J.; Hua, H. M.; Wang, H. F.; Pei, Y. H. Bioorg. Med. Chem. Lett. 2016, 26, 346−350. (20) Haritakun, R.; Rachtawee, P.; Chanthaket, R.; Boonyuen, N.; Isaka, M. Chem. Pharm. Bull. 2010, 58, 1545−1548. (21) Ma, X.; Zhu, T.; Gu, Q.; Xi, R.; Wang, W.; Li, D. J. Ocean Univ. China 2014, 13, 1067−1070. (22) Belofsky, G.; Kolaczkowski, M.; Adams, E.; Schreiber, J.; Eisenberg, V.; Coleman, C. M.; Zou, Y.; Ferreira, D. J. Nat. Prod. 2013, 76, 915−925. (23) Wang, W.; Kim, H.; Nam, S. J.; Rho, B. J.; Kang, H. J. Nat. Prod. 2012, 75, 2049−2054. (24) Kim, S. H.; Kim, K. H.; Lee, H. J.; Kim, J. N. Tetrahedron Lett. 2013, 54, 329−334.

2981

DOI: 10.1021/acs.jnatprod.6b00766 J. Nat. Prod. 2016, 79, 2977−2981