Asperimides A–D, anti-inflammatory aromatic butenolides from a tropical endophytic fungus Aspergillus terreus

Asperimides A–D, anti-inflammatory aromatic butenolides from a tropical endophytic fungus Aspergillus terreus

Fitoterapia 131 (2018) 50–54 Contents lists available at ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote Asperimides A–D,...

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Fitoterapia 131 (2018) 50–54

Contents lists available at ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Asperimides A–D, anti-inflammatory aromatic butenolides from a tropical endophytic fungus Aspergillus terreus

T



Guangfeng Liaoa,b,c, Ping Wua,b, Jinghua Xuea,b, Lan Liud, Hanxiang Lia,b, , Xiaoyi Weia,b a

Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China c University of Chinese Academy of Sciences, Beijing 100049, China d School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510006, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Butenolide Maleimide Anti-inflammatory Endophytic fungus

Four new aromatic butenolides, asperimides A–D (1–4), together with a known analogue, butyrolactone I (5), were isolated from solid cultures of a tropical endophytic fungus Aspergillus terreus. The structures of these compounds were elucidated on the basis of spectroscopic methods and electronic circular dichroism calculations. Compounds 1–4 represent the first examples of butenolides with a maleimide core isolated from Aspergillus sp. Inhibitory effects of the isolated compounds on nitric oxide production were investigated in lipopolysaccaride (LPS)-mediated RAW 264.7 cells. Compounds 3 and 4 exhibited potent anti-inflammatory with IC50 values of 0.78 ± 0.06 and 1.26 ± 0.11 μM, respectively.

1. Introduction

2. Experimental

Fungi have been recognized as a rich source of new bioactive products. The unprecedented structural diversity and potent biological activity from fungal secondary metabolites remain powerful forces driving pharmaceutical discovery [1–4]. Aspergillus species are one of the major contributors to the bioactive substances of fungal origin [5,6]. As characteristic metabolites of Aspergillus sp., aromatic butenolides exhibit diverse biological activities such as antibacterial [7], cytotoxic [8,9], anti-inflammatory [10], and antioxidant activities [11]. In our ongoing search for novel bioactive agents from fungi [12–15], we investigated the chemical components of the fermentation extract of the strain A. terreus SC1550, which was isolated from a tropical plant Suriana maritima L. Four maleimide-containing butenolides, asperimides A–D (1–4), along with a known analogue, butyrolactone I (5), were obtained from the solid substrate fermentation extract. Compounds 1–5 were tested for their inhibitory effects on nitric oxide production in lipopolysaccharide (LPS)-induced macrophages. Herein, the details of chemical and biological characterization of the isolated compounds are reported.

2.1. General



Optical rotations were obtained on a Perkin-Elmer 343 spectropolarimeter (Perkin-Elmer, Waltham, MA, USA). UV measurements were performed with a Perkin Elmer Lambda 650 UV/vis spectrometer (Perkin-Elmer, Waltham, MA, USA). ECD data were collected by a Jasco J-810 CD spectrometer (JASCO, Tokyo, Japan). IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. 1H NMR (500 MHz), 13 C NMR (125 MHz), and 2D NMR spectra were recorded on a Bruker AV-600 instrument (Bruker, Karlsruhe, Germany) with residual solvent peaks as references. ESIMS data were obtained on an MDS SCIEX API 2000 LC/MS instrument (SCIEX, Toronto, Canada). HRESIMS data were obtained on a Bruker Bio TOF IIIQ mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Preparative HPLC was performed with an HPLC system equipped with a Shimadzu LC-6 CE pump (Shimadzu, Kyoto, Japan) using a YMC-pack ODS-A column (5 μm, 10 × 250 mm, YMC, Kyoto, Japan). For column chromatography, silica gel 60 (100–200 mesh, Qingdao Marine Chemical Ltd., Qingdao, China) and YMC ODS (75 μm, YMC, Kyoto, Japan) were used. TLC was performed using HSGF254 silica gel plates (Yantai Jiangyou Silica Gel Development Co. Ltd., Yantai, China).

Corresponding author. E-mail address: [email protected] (H. Li).

https://doi.org/10.1016/j.fitote.2018.10.011 Received 13 July 2018; Received in revised form 6 October 2018; Accepted 7 October 2018 Available online 09 October 2018 0367-326X/ © 2018 Elsevier B.V. All rights reserved.

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2.2. Fungal material

Table 2 13 C NMR Data (125 MHz) in MeOD of asperimides A–D (1–4).

The fungus strain SC1550 in this study was isolated from fresh, healthy leaves of S. maritima L., which were collected from Yongxing Island, South China Sea, China. The fungus was identified as A. terreus by DNA amplification and sequencing of the ITS region. The ITS sequence data have been registered in GenBank (accession no. MH339744). A voucher strain was deposited in Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences. 2.3. Fermentation, extraction and isolation The fungus A. terreus SC1550 was fermented on autoclaved wheat grains solid-substrate medium (60 Erlenmeyer flasks, each containing 50 g of wheat grains and 50 mL of distilled water) for 28 days at 25 °C. The obtained mycelia solid culture was extracted with EtOAc three times at room temperature. The resultant extract was suspended in H2O and degreased with petroleum ether. The EtOAc-soluble fraction (40.0 g) was separated into ten fractions (A − J) by silica gel column chromatography (CC) using petroleum ether-EtOAc mixtures of increasing polarities (90:10, 70:30, 50:50) and CHCl3-MeOH mixtures of increasing polarities (99:1, 95:5, 90:10, 85:15). Fraction D (4.0 g), was separated by ODS CC using aqueous MeOH (40–80%) followed by Sephadex LH-20 CC and semipreparative HPLC using 56% aqueous CH3CN to afford 2 (8.0 mg, tR = 17.2 min). Fraction E (12.0 g), was separated by ODS CC using aqueous MeOH (40–60%) followed by semipreparative HPLC using 50% aqueous CH3CN to afford 1 (10.0 mg, tR = 30.0 min) and 5 (32.4 mg, tR = 41.5 min). Fraction F (9.0 g), was separated by silica gel CC using CHCl3-MeOH mixtures of increasing polarities (100:1–97:3) and followed by Sephadex LH-20 CC, subfraction F5 (4.0 g), was separated by Sephadex LH-20 CC using MeOH and preparative HPLC using 33% aqueous CH3CN to afford 4 (20.0 mg, tR = 13.8 min) and 3 (10.0 mg, tR = 13.9 min).

Asperimide D (4)

Position

δH, mult. J in Hz

δH, mult. J in Hz

δH, mult. J in Hz

δH, mult. J in Hz

6 2′(6′) 3′(5′) 2” 5” 6”

3.76, 7.43, 6.83, 6.83, 6.65, 6.82,

s d (8.8) d (8.8) d (2.3) d (8.2) dd (8.2,

3.79, 7.42, 6.83, 6.85, 6.64, 6.87,

s d (8.6) d (8.6) br s d (8.3) br d (8.3)

3.80, s 7.43, d (8.7) 6.82, d (8.7) 6.96, d (1.9) 6.61, d (8.3) 6.86, dd (8.3, 1.9)

7”

3.21, d (7.4)

3.79, 7.43, 6.83, 6.79, 6.61, 6.88, 2.3) 6.27,

d (9.8)

2.93, 5.3) 2.64, 7.2) 3.72, 5.3) 1.29, 1.22,

dd (16.7,

dd (7.2,

3.12, dd (15.3, 8.6) 3.07, dd (15.3, 9.4) 4.54, dd (9.4, 8.6)

s s

1.23, s 1.19, s

s d d s d d

(8.7) (8.7) (7.7) (7.7)

8”

5.24, m

5.62, d (9.8)

10” 11”

1.71, s 1.64, s

1.36, s 1.36, s

dd (16.7,

Asperimide D (4)

Position

δC, mult.

δC, mult.

δC, mult.

δC, mult.

2 3 4 5 6 1’ 2′(6′) 3′(5′) 4’ 1” 2” 3” 4” 5” 6” 7” 8” 9” 10” 11”

174.1, C 139.4, C 138.4, C 174.7, C 29.5, CH2 121.5, C 132.4, CH 116.4, CH 160.3, C 129.6, C 130.3, CH 129.4, C 154.7, C 116.0, CH 127.5, CH 28.9, CH2 123.7, CH 133.3, C 17.8, CH3 25.9, CH3

173.9, C 139.9, C 137.9, C 174.6, C 29.5, CH2 121.5, C 132.4, CH 116.4, CH 160.4, C 131.2, C 127.2, CH 122.9, C 152.9, C 117.4, CH 129.8, CH 123.2, CH 132.2, CH 77.2, C 28.1, CH3 28.1, CH3

174.0, C 139.8, C 138.0, C 174.7, C 29.5, CH2 121.4, C 132.4, CH 116.4, CH 160.4, C 130.5, C 130.6, CH 121.4, C 153.0, C 118.1, CH 128.4, CH 32.1, CH2 70.4, CH 78.0, C 25.8, CH3 21.2, CH3

174.0, C 139.7, C 138.2, C 174.7, C 29.7, CH2 121.5, C 132.4, CH 116.4, CH 160.4, C 130.8, C 125.8, CH 129.1, C 160.1, C 109.8, CH 128.8, CH 31.5, CH2 90.5, CH 72.5, C 25.3, CH3 25.2, CH3

2.3.4. Asperimide D (4) Light yellow gum; [α]20 D − 26.8 (c 0.38, MeOH); UV (MeOH) λmax (log ε): 199 (3.79), 231 (3.36), 284 (2.76), 360 (2.69); IR νmax 3333, 2918, 1707, 1607, 1489, 1350, 1246, 1016, 802 and 667 cm−1; CD (MeOH) λmax (Δε) 230 (−1.75); 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS: m/z 402.1331 [M + Na]+ (calcd for C22H21NO5Na, 402.1317).

Table 1 1 H NMR Data (500 MHz) in MeOD of asperimides A–D (1–4). Asperimide C (3)

Asperimide C (3)

2.3.3. Asperimide C (3) Light yellow gum; [α]20 D + 4.4 (c 0.62, MeOH); UV (MeOH) λmax (log ε): 196 (3.83), 230 (3.40), 276 (2.76), 365 (2.75); IR νmax 3331, 2916, 1705, 1607, 1497, 1348, 1259, 1244, 1020, 804 and 659 cm−1; CD (MeOH) λmax (Δε) 232 (+ 1.10); 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS: m/z 402.1313 [M + Na]+ (calcd for C22H21NO5Na, 402.1317).

2.3.2. Asperimide B (2) Light yellow gum; UV (MeOH) λmax (log ε): 224 (3.95), 325 (3.14),

Asperimide B (2)

Asperimide B (2)

357(3.14); IR νmax 3308, 1705, 1607, 1516, 1489, 1338, 1211, 1175, 841 and 763 cm−1; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS: m/z 384.1211 [M + Na]+ (calcd for C22H19O4Na, 384.1212).

2.3.1. Asperimide A (1) Light yellow gum; UV (MeOH) λmax (log ε): 229 (3.69), 278 (3.08), 360 (3.04); IR νmax 3300, 1703, 1607, 1514, 1435, 1348, 1267, 1175, 841, 816 and 766 cm−1; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS: m/z 386.1367 [M + Na]+ (calcd for C22H21NO4Na, 386.1368).

Asperimide A (1)

Asperimide A (1)

2.4. Biological assays RAW264.7 cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, People's Republic of China). Cell maintenance, experimental procedures, the determination of data for the inhibition of the production of NO, and the viability assay were performed according to a modified literature procedure [16]. RAW 264.7 cells were seeded in 96-well plates (Nunc) at a density of 1 × 105 cells per well and incubated overnight. Then the cells were treated with LPS (1 μg/mL) and various concentrations of the compounds for 24 h. Subsequently, 50 μL nitric oxide detection regent I and 50 μL nitric oxide detection reagent II were added to each well, respectively. The absorbance was measured at 540 nm with an Infinite M200 PRO microplate reader (TECAN). The IC50 values were determined using Origin 8 Pro software from experiments performed in triplicate. Indomethacin (IC50 value of 37.5 ± 1.6 μM) was used as a positive control. All the tested compounds were prepared as stock solutions in DMSO, and the final solvent concentration was less than 0.2% in all assays (Fig. 1). 51

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Fig. 1. Structure of compounds 1–5.

Fig. 2. Key 1He1H COSY (bold lines) and HMBC (arrows) correlations of 1–4.

Fig. 4. Comparison between the measured and CAM-B3LYP/TZVP/PCM (MeOH) calculated ECD spectra of 4.

TDDFT calculations were performed using the hybrid CAM-B3LYP [20] and M06-2× functionals, and Ahlrichs' basis set TZVP (triple zeta valence plus polarization) [21]. The number of excited states per each molecule was 42 for both 3 and 4. In all DFT and TDDFT calculations, solvent effects were taken into account using polarizable continuum model (PCM) for MeOH. The ECD spectra were generated by the program SpecDis [22] using a Gaussian band shape from dipole-length dipolar and rotational strengths. Equilibrium population of each conformer at 298.15 K was calculated from its relative free energies using Boltzmann statistics. The calculated spectra were generated from the low-energy conformers according to the Boltzmann weighting of each conformer in MeOH solution. Fig. 3. Comparison between the measured and CAM-B3LYP/TZVP/PCM (MeOH) calculated ECD spectra of 3.

3. Results and discussion Compound 1 was obtained as a light yellow gum. Its molecular formula was determined to be C22H21NO4 on the basis of HRESIMS. The 1 H NMR spectrum indicated the signals of a para-substituted phenol (A2B2 system), a 3,4-disubstituted benzyl ring, and an isoprenyl group at δH 5.24 (1H, m), 3.21 (2H, d, 7.4 Hz), 1.71 (3H, s), and 1.64 (3H, s). Inspection of the 13C NMR spectrum showed 20 well-resolved resonance peaks, of which two carbonyl carbons (δC 174.7 and 174.1) and two olefinic carbons (δC 139.4 and 138.4) were characteristic of a maleimide nucleus [23,24]. The isoprenyl unit was connected to the benzyl ring at C-3″ as deduced from HMBC correlations between H2–7″ to C-3″, C-4″ and C-2″ (Fig. 2), thus establishing the 4-hydroxy-3-isoprenyl benzyl moiety. In turn, HMBC spectrum showed cross peaks from the benzyl methylene protons H2–6 to C-3/C-4/C-5, indicating that the 4-hydroxy-3-isoprenyl benzyl unit was connected to the C-4 of

2.5. Computational methods Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with Spartan’14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian09 program package [17], respectively. MMFF conformational search generated low-energy conformers within a 10 kcal/mol energy window were subjected to geometry optimization using the DFT method at the B3LYP/def2-SVP level of theory. Frequency calculations were run at the same level to verify that each optimized conformer was a true minimum and to estimate their relative thermal free energies (ΔG) at 298.15 K. The optimized minima within the relative energies of 4.0 kcal/mol were subjected to the higher level of energy calculations at the M06-2× [18]/def2-TZVP [19] level. The 52

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Fig. 5. Proposed biosynthetic pathway for compounds 1–4.

Therefore, the absolute configuration of 4 was deduced to be 8”R. In addition, the known compound 5 was identified as butyrolactone I by comparing the measured spectroscopic data with the reported in the literature [25]. Compounds 1–5 were investigated for their inhibitory activities against the LPS-activated production of NO in RAW264.7 cells using the Griess assay with indomethacin as a positive control (IC50 = 37.5 ± 1.6 μM). The effects of compounds on cell proliferation/viability were determined using MTT method, and none of the test compounds exhibited cytotoxicity at their effective concentrations. Compounds 3, 4 and 5 showed strong inhibitory effects on the production of NO, with IC50 values of 0.78 ± 0.06, 1.26 ± 0.11, and 24.2 ± 1.6 μM, respectively. Further studies are required to clarify the underlying mechanism of the active compounds. Aspergillus sp. are well known for the production of butenolides. Since the first report on the chemical structure of butyrolactone I in 1977 [25], a number of related phenyl- and benzyl-disubstituted butenolides have been described [8–11,16]. This class of butenolides, biogenetically derived from tyrosine and/or phenyl alanine, can be classified into three types in accordance with the substituted pattern of the lactone core, which are 2,3-, 3,4-, and 2,4-disubstituted ones. Presumably, the biosynthesis of asperimides (1–4) proceeds in a manner similar to that of other buteolides. It is interesting to note that compounds 1–4 differ from the precedent butenolides from Aspergillus sp. by having a maleimide core. Based on these findings, a biosynthetic pathway for 1–4 starting from tyrosine is proposed as shown in Fig. 5 [26,27].The new natural buteolide containing-maleimide identified in this research expand the chemical space and biological diversity of aromatic buteolides.

the maleimide core via C-6. In addition, the 4-hydroxyl phenyl ring was attached to C-3 of the maleimide group, due to the HMBC correlations from H-2′(6′) to C-3. This completed the gross structure of compound 1, for which we propose the trivial name asperimide A. The molecular formula C22H19NO4, implying 14 unsaturation degrees, was assigned to asperimide B (2) by HRESIMS. The additional degree of unsaturation, relative to 1, and the shift of resonances of the prenyled group indicated cyclization of the isoprenyl chain with one of the ortho hydroxyl groups, forming a 2,2-dimethyl-2H-pyran moiety. These observations were supported by COSY correlations between two trans olefinic protons H-7”/H-8″ and HMBC cross-peaks of H-7″ with C2”/C-3”/C-4″ (Fig. 2). The 1H NMR spectrum of asperimide C (3) (C22H21NO5 by HRESIMS) clearly suggested that this compound shared the same carbon skeleton as asperimide B (2), except that the olefinic double bond in 2,2-dimethyl-2H-pyran ring was replaced by resonances for a methylene and an oxymethine. The inference was confirmed by the chemical shift of C-8″ (δH/δC 3.72/70.4) and HMBC correlations from H2–7″ to C-2”/C-3”/C-4″ and from H-8″ to C-3″ (Fig. 2), suggesting that 3 is the hydration product of 2. The absolute configuration of 3 was deduced by comparison of the experimental and calculated ECD spectra (Fig. 3). The experimental CD spectrum of 3 was nearly identical to the calculated ECD spectrum of 8”S-3, both showing negative Cotton effects at 230 nm. Therefore, the absolute configuration of 3 was assigned to be 8”S. HRESIMS indicated that asperimide D (4) possessed the same molecular formula as 3. Similar reasoning was applied to assign its structure. A dihydrofuran moiety instead of the dihydropyran ring was indicated by the significant downfield shift observed for the oxymethine NMR resonances in 4 (δH/δC 4.54/90.5), relative to the corresponding signals in 3 (δH/δC 3.72/70.4). Accordingly, HMBC correlation was also experienced from H-8″ to C-4″ (Fig. 2). The absolute configuration of 4 was deduced by ECD/EDDFT calculations using the procedure as described for compound 3. It can be seen that the calculated CD spectrum provided excellent fits with the experimental spectrum (Fig. 4).

Conflict of interest No potential conflicts of interest relevant to this article were reported. 53

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Acknowledgments

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We gratefully acknowledge financial support from NNSFC grant [grant numbers 21502197 and 81172942] and Science and Technology Planning Project of Guangdong Province, China [grant number 2015A020211023]. We gratefully acknowledge support from the Guangzhou Branch of the Supercomputing Center of Chinese Academy of Sciences, and thank Ai-Jun Sun and Yun Zhang, South China Sea Institute of Oceanology, Chinese Academy of Sciences, for the measurement of HRESIMS. Appendix B. Supplementary data Supplementary data (1D, 2D NMR, UV, IR, LRESIMS and HRESIMS spectra of compounds 1–4) to this article can be found online at: https://doi.org/10.1016/j.fitote.2018.10.011. References [1] B. Schulz, C. Boyle, S. Draeger, A.-K. Römmert, K. Krohn, Endophytic fungi: a source of novel biologically active secondary metabolites, Mycol. Res. 106 (2002) 996–1004. [2] M.E. Rateb, R. Ebel, Secondary metabolites of fungi from marine habitats, Nat. Prod. Rep. 28 (2011) 290–344. [3] A.L. Harvey, R. Edrada-Ebel, R.J. Quinn, The re-emergence of natural products for drug discovery in the genomics era, Nat. Rev. Drug Discov. 14 (2015) 111. [4] A. Du Toit, Fungal biology: a key regulator of secondary metabolites, Nat. Rev. Microbiol. 14 (2016) 727. [5] J.C. Frisvad, T.O. Larsen, Chemodiversity in the genus Aspergillus, Appl. Microbiol. Biotechnol. 99 (2015) 7859–7877. [6] Y.M. Lee, M.J. Kim, H. Li, P. Zhang, B. Bao, K.J. Lee, J.H. Jung, Marine-derived Aspergillus species as a source of bioactive secondary metabolites, Mar. Biotechnol. 15 (2013) 499–519. [7] S.R. Ibrahim, E.S. Elkhayat, G.A. Mohamed, A.I. Khedr, M.A. Fouad, M.H. Kotb, S.A. Ross, Aspernolides F and G, new butyrolactones from the endophytic fungus Aspergillus terreus, Phytochem. Lett. 14 (2015) 84–90. [8] W. Gu, C. Qiao, Furandiones from an endophytic Aspergillus terreus residing in Malus halliana, Chem. Pharm. Bull. 60 (2012) 1474–1477. [9] K. Rao, A. Sadhukhan, M. Veerender, V. Ravikumar, E. Mohan, S. Dhanvantri, M. Sitaramkumar, J.M. Babu, K. Vyas, G.O. Reddy, Butyrolactones from Aspergillus terreus, Chem. Pharm. Bull. 48 (2000) 559–562. [10] F. Guo, Z. Li, X. Xu, K. Wang, M. Shao, F. Zhao, H. Wang, H. Hua, Y. Pei, J. Bai, Butenolide derivatives from the plant endophytic fungus Aspergillus terreus, Fitoterapia 113 (2016) 44–50. [11] L.-J. Li, T.-X. Li, L.-Y. Kong, M.-H. Yang, Antioxidant aromatic butenolides from an insect-associated Aspergillus iizukae, Phytochem. Lett. 16 (2016) 134–140. [12] Y. Fu, P. Wu, J. Xue, X. Wei, Cytotoxic and antibacterial quinone sesquiterpenes from a Myrothecium fungus, J. Nat. Prod. 77 (2014) 1791–1799. [13] J. Xue, P. Wu, L. Xu, X. Wei, Penicillitone, a potent in vitro anti-inflammatory and cytotoxic rearranged sterol with an unusual tetracycle core produced by Penicillium purpurogenum, Org. Lett. 16 (2014) 1518–1521.

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