Avertoxins A–D, Prenyl Asteltoxin Derivatives from Aspergillus

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Avertoxins A−D, Prenyl Asteltoxin Derivatives from Aspergillus versicolor Y10, an Endophytic Fungus of Huperzia serrata Mingzi Wang,† Mingwei Sun,‡ Huilin Hao,‡ and Chunhua Lu*,‡ †

Engineering Research Center of Industrial Microbiology (Ministry of Education), College of Life Sciences, Fujian Normal University, Fuzhou, Fujian 350117, People’s Republic of China ‡ Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, People’s Republic of China S Supporting Information *

ABSTRACT: Aspergillus versicolor Y10 is an endophytic fungus isolated from Huperzia serrata, which showed inhibitory activity against acetylcholinesterase. An investigation of the chemical constituents of Y10 led to the isolation of four new prenylated asteltoxin derivatives, named avertoxins A−D (2−5), together with the known mycotoxin asteltoxin (1). In the present study, we report structure elucidation for 2−5 and the revised NMR assignments for asteltoxin and demonstrated that avertoxin B (3) is an active inhibitor against human acetylcholinesterase with the IC50 value of 14.9 μM (huperzine A as the positive control had an IC50 of 0.6 μM). In addition, the cytotoxicity of asteltoxin (1) and avertoxins A−D (2−5) against MDA-MB-231, HCT116, and HeLa cell lines was evaluated.

A

lzheimer’s disease (AD) is a common form of dementia that is characterized by the deposition of amyloids in affected neurons and a cholinergic neurotransmission deficit in the brain.1 The current therapeutic intervention for AD is primarily based on the inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level.2 Huperzine A is a potent, reversible, and selective AChE inhibitor isolated from the traditional Chinese medicine Huperzia serrate.3,4 To investigate the potential of endophytic fungal sources isolated from H. serrata, we assayed their inhibitory activity against AChE. The extract of Aspergillus versicolor Y10 strain isolated from the leaves of H. serrata showed moderate activity against AChE, with an IC50 of 394 μg/mL. This strain was selected for further fermentation and chemical analysis. The Y10 strain was cultured on 5 L of potato-dextrose-agar (PDA) medium, and an extract was subjected to bioassay-guided fractionation, which resulted in the isolation of four new polyenic alpha-pyrone mycotoxins, named avertoxins A−D (2−5), together with the known asteltoxin. Their structures were elucidated by extensive spectroscopic analysis as well as by comparison with previously reported data. Their inhibitory activity against AChE and cytotoxicity were evaluated. Solid-state fermentation (5 L) of the Y10 strain was performed in PDA media incubated at 28 °C for 14 d. The cultured agar was chopped, diced, and extracted for overnight with EtOAc at room temperature. The extract obtained after the removal of solvents under vacuum was subjected to medium-pressure liquid chromatography (MPLC), column chromatography over Sephadex LH-20, and silica gel to yield compounds 1−5. © XXXX American Chemical Society and American Society of Pharmacognosy

Compound 1 was obtained as a light yellow, amorphous powder. Its HRESIMS revealed a quasi-molecular ion peak at m/z 419.2074 [M + H]+ suggesting the molecular formula C23H30O7. The UV absorption at 385 nm, near the visible, is Received: July 7, 2015

A

DOI: 10.1021/acs.jnatprod.5b00600 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 2−5a 2

a

position

δC

1 2 3 4 4a 5 5a 6 7 8 9 10 11 12 13 14 15 16 16a 17 18 19 1′ 2′ 3′ 4′ 5′ MeO-4′ HO-4 HO-7

11.8 22.5 90.1 80.9 18.3 62.6 16.6 113.0 80.2 84.9 134.0 133.3 138.0 132.7 135.6 121.1 154.8 109.0 9.1 170.1 90.1 162.7 66.9 119.0 140.0 25.8 18.3

δH, mult (J, Hz) 0.98, t (7.5) 1.60−1.45, m 4.34, dd (3.6, 9.1) 1.36, s 1.18, 5.16, 3.84, 4.65, 6.07, 6.51, 6.69, 6.55, 7.10, 6.65,

s s dd (3.2, 5.3) br d (6.6) dd (9 6.6, 15.2) dd (11.2, 15.0) dd (11.0, 15.5) dd (11.1, 16.0) dd (11.0, 14.9) d (14.5)

1.98, s 5.53, s 4.63, br s 5.51, tt (0.1, 6.6) 1.80, s 1.77, s 3.37, s 4.03, d (5.4)

3 δC 11.8 22.5 90.2 81.0 18.4 62.6 16.6 113.1 80.3 84.9 134.1 133.3 138.2 132.7 135.9 121.0 155.1 108.7 9.1 169.8 90.5 162.6 66.9 135.2 131.7 167.8 13.2 52.3

4

δH, mult (J, Hz)

δC

0.98, t (7.5) 1.62−1.44, m 4.34, dd (3.6, 9.1) 1.36, s 1.18, 5.16, 3.84, 4.66, 6.08, 6.51, 6.70, 6.55, 7.12, 6.67,

s s dd (3.2, 5.3) dd (2.7, 6.4) dd (6.6, 14.8) dd (11.2, 15.0) dd (overlapped) dd (11.1, 16.0) dd (11.2, 15.1) d (14.5)

2.02, s 5.59, s 4.93, dd (0.6, 5.6) 6.88, dt (1.3, 5.6)

1.95, 3.75, 3.36, 4.02,

d (1.1) s s d (5.4)

13.5 90.5 81.0 17.9 62.3 16.8 113.0 80.2 84.8 134.0 133.3 138.2 132.7 135.8 121.0 155.1 108.7 9.1 169.8 90.5 162.6 66.9 135.3 131.7 167.8 13.3 52.3

δH, mult (J, Hz) 1.08, d (6.4) 4.56, q (6.4) 1.36, s 1.19, 5.16, 3.84, 4.65, 6.07, 6.51, 6.68, 6.56, 7.12, 6.67,

s s dd (2.6, 5.4) m dd (6.6, 15.2 dd (overlapped) d (14.7) dd (overlapped) dd (11.1, 14.9) d (14.5)

2.09, s 5.63, s 4.94, d (5.6) 6.87, dt (1.3, 5.6)

1.95, 3.75, 3.38, 4.05,

d (1.1) s s d (5.4)

5 δC

δH, mult (J, Hz)

11.8 22.5 90.3 81.0 18.4 62.6 16.6 113.1 80.3 84.9 134.1 133.3 138.2 132.7 135.8 120.8 157.4 105.2 8.9 169.7 110.1 160.0 44.1 92.3 14.8 25.7 20.4

0.98, t (7.5) 1.60−1.45, m 4.34, dd (3.6, 9.1) 1.36, s 1.18, 5.16, 3.84, 4.65, 6.08, 6.51, 6.71, 6.54, 7.11, 6.60,

s s dd (3.0, 5.4) dd (2.7, 6.4) dd (6.4, 15.2) dd (overlapped) dd (10.5, 14.9) dd (overlapped) dd (10.4, 15.0) d (15.0)

2.00, s

4.60, 1.40, 1.35, 1.16,

q (6.6) d (6.6) s s

3.37, s 4.02, d (5.4)

Recorded in acetone-d6 (1H NMR for 400 MHz, 13C NMR for 100 MHz).

Figure 1. Selected NOE correlations for compounds 2, 3, and 5.

molecular formula of 2 was established as C27H36O7 based on the HRESIMS data. The 1H and 13C NMR, HSQC, and HMBC spectroscopic data showed the presence of a 2isopentenyloxyl group at δH 1.77 (3H, s, H-5′), 1.80 (3H, s, H4′), 4.63 (br s, H-1′), and 5.51 (tt, J = 0.1, 6.6, H-2′) and δC 66.9 (C-1′), 119.0 (C-2′), 140.0 (C-3′), 25.8 (C-4′), and 18.3 (C-5′) (Table 1). The remaining NMR signals were attributed to four methyl, one methylene, 11 methine (four being oxygenated), and six quaternary carbons (C-4, C-5, C-15, C-16, C-17, and C-19), which were similar to those of asteltoxin (1). The distinct difference between compounds 2 and 1 is that the OMe-17 of asteltoxin [δH 3.91 (3H, s), δC 57.0] (Table S1) was replaced by a 2-isopentenyl oxyl group, which was further confirmed by the HMBC correlations from H-1′ to C-17, C-2′, and C-3′, from H-4′ to C-5′, C-2′, and C-3′, and from H-5′ to C-4′. The relative configurations of 2 were determined on the basis of the NOESY experiments. The NOE correlations of H-

characteristic of a yellow color and indicates the presence of conjugated double bonds. The IR absorptions at 1695, 1622, and 1540 cm−1 (KBr) indicate the presence of an α-pyrone unit. The 1H and 13C NMR spectroscopic data of 1 are similar to those of asteltoxin.5 However, the NMR assignments reported in literature should be exchanged between C-4a and C-5a, C-9 and C-14, and C-17 and C-19, respectively (Table S1). The key HMBC correlations from OMe-17 to C-17, from H-4a to C-3, C-4, and C-5, from H-5a to C-4, C-5, C-6, and C7, and from H-9 to C-8, C-10, and C-11 further collaborated those changes. Additionally, these NMR assignments are consistent with that for asteltoxin C.6 It noteworthy that, in α-pyrones, the 13C NMR chemical shifts of the α-positions (carbonyl) usually are more upfield than that of the γ-positions (hydroxy).7 Compounds 2−5 were all obtained as light yellow powders and showed similar IR spectra to that of asteltoxin. The B

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5a/H-6/H-7/H-8, H-3/H-4a, H-1′/H-5′, and H-2′/H-4′ suggested that C-4a, HO-7, and C-8 were α-oriented and that C-2, HO-4, C-5a, and H-6 were β-oriented, the same as those of asteltoxin determined by single-crystal X-ray diffraction.5 This assignment was supported by the similar optical rotations of 2 and 1. The geometry of the 2-isopentenyl oxyl group at C17 was determined on the basis of NOE correlations between δH 4.63 (H-1′) and 1.77 (H-5′) and between δH 5.51 (H-2′) and 1.80 (H-4′) (Figure 1). The molecular formula of compound 3 was established as C28H36O9 by the HRESIMS data. Interpretation of the 1H and 13 C NMR, HSQC, and HMBC spectroscopic data showed the presence of a 3-methyl-3-methoxycarbonyl-2-isopropenyloxyl group [(δH 1.95 (3H, d, J = 1.1 Hz, H-5′), 6.88 (dt, J = 1.3, 5.6 Hz, H-2′), 4.93 (dd, J = 0.6, 5.6 Hz, H-1′), 3.75 (3H, s, MeO4′); δC 66.9 (C-1′), 135.2 (C-2′), 131.7 (C-3′), 167.8 (C-4′), 13.2 (C-5′), 52.3 (MeO-4′)] (Table 1). The remaining NMR signals were similar to those of 2 (Table 1). The only difference is that the Me-4′ in 2 was replaced by a methoxycarbonyl group in 3, which was confirmed by the HMBC correlations from H5′ to C-2′, C-3′, and C-4′ and from MeO-4′ to C-4′. The molecular formula of compound 4 was established as C27H34O9 by HRESIMS data. The 1H and 13C NMR data of 4 were similar to those of 3 (Table 1). The only difference is that the ethyl group at C-3 [δH 0.98 (t, J = 7.5 Hz, 3H, H-1) and 1.62−1.44 (m, 2H, H-2); δC 11.8 (C-1) and 22.5 (C-2)] in 3 was replaced by a methyl group (δH 1.08, d, J = 6.4 Hz; δC 13.5) in 4. The molecular formula of compound 5 was established as C27H36O7 based on the HRESIMS. The presence of a 2,3dihydro-2,3,3-trimethylfuran ring was evidenced by the characteristic NMR signals at δH 1.40 (3H, d, J = 6.6 Hz, H3′), 1.35 (3H, s, H-4′), 1.16 (3H, s, H-5′), and 4.60 (1H, q, J = 6.6 Hz, H-2′) and δC 44.1 (C-1′), 92.3 (C-2′), 14.8 (C-3′), 25.7 (C-4′), and 20.4 (C-5′) (Table 1) and the HMBC correlations from Me-3′ to C-2′ and C-3′, from Me-4′ to C-1′, C-2′, and C5′, and from Me-5′ to C-1′, C-2′, and C-4′.8 The remaining NMR signals were similar to those of 1. Biological activities of compounds 1−5 were evaluated against human acetylcholinesterase in vitro. The results show that avertoxin B (3) is an inhibitor of AChE, with an IC50 value of 14.9 μM, with huperzine A as the positive control (IC50 0.6 μM), which indicated that compound 3 might be the main constituent responsible for activity in the extract. In addition, compounds 1−5 were evaluated for their cytotoxicity against human tumor MDA-MB-231, HCT116, and HeLa cell lines. Cells were treated with the compounds for 72 h in DEME medium supplemented with 10% fetal bovine serum (FBS), and cell viability was evaluated by a sulforhodamine B (SRB) assay. Interestingly, only compounds 3 and 4 showed activity against HCT116 and HeLa cell lines, with IC50 values about 10 μM (Table 2). Asteltoxin (1) and structurally related verrucosidins,9 citreoviridins,10,11 and aurovertins12−14 are a family of polyene α-pyrone-type polyketide mycotoxins.15 Recently, asteltoxins C and D, with antiproliferative activity against NIAS-SL64 cells, were reported.6 A new aurovertin, I, was isolated from a rootknot nematode parasitic fungus, Pochonia chlamydosporia; it was also shown that aurovertins D and F were toxic to the freeliving nematode Panagrellus redivevus.16 This family has potent activity against ATP synthesis and ATP hydrolysis.11,17,18 Avertoxins A−D (2−5) are novel derivatives of asteltoxin. Specifically, 2, 3, and 5 are O-prenyl and C-prenyl asteltoxins,

Table 2. IC50 Values (μM) of Compounds 1−5 Tested against Human Tumor Cell Lines compound

MDA-MB-231

HCT116

HeLa

1 2 3 4 5 VP16

>50 37 35 28 >50 4.2

>50 44 9 21 50 26.1

>50 48 >50 11 48 3.2

respectively. The structural difference between compounds 3 and 4 is the replacement of the ethyl group with a methyl group. The feeding experiments results showed that asteltoxin could be formed via two biosynthesis pathways.15,19 Pathway A involves the formation of a C20-polyketide chain with an acetate initiating unit and nine malonate units, followed by the loss of the starter unit, through a retro-Claisen cleavage, and the origin of the methyl group at C-1 was incorporated from methionine. Pathway B involves a C19-polyketide chain derived from a propionate chain-initiating unit and eight chain elongation units of malonate. Our isolation of 4 without the methyl group at C-1 provides further evidence that the C-1 methyl group is derived from methionine with post-PKS modification steps in pathway A. Therefore, it is more reasonable biosynthetically to have 4 as a nonmethylated version of 3. The anti-AChE activity of 3 is worthy of further study because it represents a novel scaffold among the many known AChE inhibitors.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with an Anton Paar MCP200 polarimeter. UV spectra were recorded in MeOH on a TU-1800. IR spectra were recorded on a Nicolet 5700 Fourier transform infrared spectroscopy (FT-IR) microscope spectrometer (FTIR Microscope Transmission). NMR spectra were recorded on a Bruker Avance DRX-400 spectrometer operating at 400 (1H) and 100 (13C) MHz. HRESIMS were carried out on an LTQ-Orbitrap XL. All solvents were analytical grade. Silica gel (200−300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, P. R. China) and Sephadex LH-20 (25−100 μm; Pharmacia Biotek, Denmark) were used for column chromatography. Thin-layer chromatography (TLC) was carried out with glass precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). Compounds were visualized under UV light and by spraying with H2SO4/EtOH (1:9, v/v) followed by heating. Pure AChE, DTNB (5,5′-dithiobis(2nitrobenzoic acid), DMSO, and SDS were purchased from Sigma. Authentic huperzine A was purchased from China National Institute for Food and Drug Control as a positive control. Acetylcholinesterase Inhibitory Assay in Vitro. In vitro AChE inhibition activity of the compounds was compared with authentic huperzine A at various concentrations. AChE inhibitory activity was carried out as the method of Mukherjee by a 96-well microplate reader based on Ellman’s method.20 Using 96-well plates, 20 μL of 1.05 mmol/L acetylthiocholine iodide (ATCh), 10 μL of sample dissolved in buffer containing not more than 10% methanol, 10 μL of 1.0 μg/mL AChE, and 40 μL of 0.02 mol/L (pH 7.2) PBS were added to the wells, which were incubated for 30 min at 37 °C, and the reaction was terminated by 4% SDS solution. Then 100 μL of 1.5 mmol/L DTNB was added, and the absorbance at 405 nm was measured. Percentage of inhibition activity was calculated by comparing the rates to the blank (10% MeOH in buffer). A range of concentrations was measured so that the IC50 value could be calculated.21 In Vitro Antiproliferative Activity. The in vitro antiproliferative activities of compounds 1−5 were assessed with the SRB assay as described previously.22,23 C

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Microbial Strains. The Y10 strain was isolated from leaves of Huperzia serrata collected from Meihua Mountain (Longyan, Fujian, China) and was identified as Aspergillus versicolor by analyzing the internal transcribed spacer regions (GenBank accession number KF381083) and morphology. Fermentation and Isolation. Fermentation was performed for 14 d at 28 °C in PDA media (5.0 L). The cultured agar was chopped, diced, and extracted three times with ethyl acetate (EtOAc) at room temperature overnight. The organic solution was collected by filtration. The combined filtrates were concentrated to remove the organic solvents, which gave the EtOAc extract (1.5 g). The extract was subjected to column chromatography (CC) over Sephadex LH-20 (130 g, acetone) to obtain Fr. 1−12. After solvent evaporation, some needle crystals appeared from Fr. 6. The mother solution (160 mg) was further purified by MPLC over RP-18 SiO2 (30 g), eluted with H2O and then a stepwise gradient of 50, 65, 85, and 100% (v/v) CH3CN in H2O, to afford Fr. 6a−6d. Fr. 6a was purified by CC (SiO2, 1.8 g) eluted with petroleum ether (PE)/acetone (4:1) to yield 1 (11 mg). Fr. 4 (7 mg) was subjected to MPLC over RP-18 SiO2 (30 g), eluted with H2O and then a stepwise gradient of 50, 65, 85, and 100% (v/v) CH3CN in H2O, to afford 2 (10 mg), 3 (10 mg), and Fr. 4a. Fr. 4a (65%-4 + 5) (20 mg) was subjected to CC (SiO2, 1.5 g) eluted with PE/acetone (15:1, 32 mL; 10:1, 44 mL; 8:1, 45 mL) to obtain 5 (2 mg). Fr. 5 (40 mg) was subjected to MPLC over RP-18 SiO2 (30 g), eluted with H2O and then a stepwise gradient of 50, 65, 85, and 100% (v/v) CH3CN in H2O, to afford 4 (2 mg). Asteltoxin (1): light yellow powder; [α]20D = +19.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε), 385 (1.36), 363 (3.47), 272 (3.47) nm; IR (KBr) νmax 3431, 2965, 2933, 1697, 1622, 1540, 1405, 1251 cm−1; 1H and 13C NMR data, Table S1; HRESIMS m/z 419.2074 [M + H]+ (calcd for C23H31O7, 419.2070). Avertoxin A (2): light yellow powder; [α]20D = +27.6 (c 0.05, MeOH); UV (MeOH) λmax (log ε), 359 (3.73), 271 (3.74) nm; IR (KBr) νmax 3431, 2967, 2929, 1694, 1622, 1537, 1384, 1241 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 473.2516 [M + H]+ and 945.4952 [2 M + H]+ (calcd for C23H37O7, 473.2539). Avertoxin B (3): light yellow powder; [α]20D = +9.75 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 359 (3.78), 272 (3.90) nm; IR (KBr) νmax 3431, 2970, 2926, 1709, 1619, 1535, 1425, 1384 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 517.2399 [M + H]+ (calcd for C28H37O9, 517.2438). Avertoxin C (4): light yellow powder; [α]20D = +26.12 (c 0.08, MeOH); UV (MeOH) λmax (log ε), 362 (3.49), 270 (3.47) nm; IR (KBr) νmax 3431, 2972, 2925, 1709, 1619, 1525, 1425 cm−1; 1H and 13 C NMR data, Table 1; HRESIMS m/z 503.2279 [M + H]+ (calcd for C27H35O9, 503.2281). Avertoxin D (5): light yellow powder; [α]20D = +68.29 (c 0.07, MeOH); UV (MeOH) λmax (log ε), 364 (3.52), 348 (3.48), 270 (3.42), 271 (3.74) nm; IR (KBr) νmax 3431, 2975, 2932, 1695, 1622, 1525 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 473.2523 [M + H]+ (calcd for C27H37O7, 473.2539).

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ACKNOWLEDGMENTS This work was financially supported by the 973 Programs (2010CB833802; 2012CB721005), the National Natural Science Foundation of China (Nos. 81373304 and 31070053), and Program for Changjiang Scholars and Innovative Research Team in University (IRT13028).

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REFERENCES

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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.5b00600. 1 H and 13C NMR assignments for 1 (Table S1) and NMR and HRESIMS spectra for 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (C.-H. Lu): +86-531-8838-2108. Fax: +86-531-88382485. E-mail: [email protected]. Notes

The authors declare no competing financial interest. D

DOI: 10.1021/acs.jnatprod.5b00600 J. Nat. Prod. XXXX, XXX, XXX−XXX