Isochromophilones from an endophytic fungus Diaporthe sp.

Introduction

The azaphilones are a structurally diverse family of natural products containing a highly oxygenated, bicyclic core and a chiral quaternary center. The name of azaphilone arose as a result of their affinity for ammonia: the pigments react with amines, such as proteins, amino acids and nucleic acids, to form red and purple vinylogous γ-pyridones due to the exchange of pyrane oxygen for nitrogen^1-3. Azaphilone compounds had a wide range of bioactivities including cytotoxic, antifungal, antimicrobial, antioxidant and antiinflammatory activities, ^4-8 and could be generally produced by Penicillium, Chaetomium and Monascus.³ In the course of our search for novel and/or bioactive metabolites from microbes lived in special niche, ^{4, 9, 10} five azaphilones including three new isochromophilones Ⅹ–Ⅻ (1–3) and two known compounds sclerotioramine (4)¹¹ and isochromophilone Ⅵ (5)¹² were isolated from the solid-substrate fermentation cultures of an endophytic fungus Diaporthe sp. The structures were identified on the basis of extensive spectroscopic methods. The cytotoxic activities of compounds (1–5) against human gastric cancer SGC-7901, colorectal carcinoma SW1116, breast adenocarcinoma MCF-7, lung adenocarcinoma epithelial A549 and melanoma A375 cells were evalutated by MTT method. Compound 1 exhibited moderate cytotoxic activities with its IC₅₀ values ranging from 14.90–35.75 μM.

Results and Discussion

Compound 1 was obtained as red needles, the molecular formula was assigned as C₂₉H₃₂ClNO₄ evidenced by its HRESIMS spectrum at m/z 494.2080 [M + H]⁺ (calcd. for C₂₉H₃₃³⁵ClNO₄, 494.2098), indicating 14 degrees of unsaturation. Compound 1 displayed similar ¹H NMR spectrum to that of known compound isochromophilone Ⅵ (5) except for a monosubstituted benzene ring signals [δ 7.26 (m, 1H), 7.32 (m, 2H), 7.10 (d, 7.0, 2H)] in 1, which suggested that the 2'-OH in isochromophilone Ⅵ (5) could be replaced by a benzene ring in compound 1. To confirm the proposed structure, extensive NMR spectra have been investigated (Figure 2). ¹H-¹H COSY correlations of H-10/H-11, and of Me-18/H-13/H-14/H-15(Me-17)/Me-16, together with the HMBC correlations from H-10 to C-12, H-11 to C-18, H-13 to C-11, C-15, C-17, C-18 indicated the presence of a 3, 5-dimethyl-1, 3-heptadiene residue. On the basis of the coupling constants of H-10, H-11, and the NOE correlation of H-11 with H-13, the configurations of these two double bonds were both determined to be E. The HMBC correlations from H-1' to C-3', H-4' to C-2', H-7' to C-3' confirmed the linkage of C-2' and the benzene ring. Moreover, the HMBC correlations of H-1 to C-1', C-2, C-4, C-5, C-8, C-9, of H-3 to C-5 and C-10, of Me-19 to C-6, C-8 indicated the presence of isoquinoline-6, 8(2H, 7H)-dione moiety, a typical group in azaphilone skeleton and the way the residues linked at. Thus, the planar structure of 1 was established.

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Compound 2, isolated as red needles, was determined to have the molecular formula C₂₇H₃₀ClNO₃ (13 degrees of unsaturation) based on an HRESIMS peak at m/z 452.1998 [M + H]⁺ (calcd. for C₂₇H₃₁³⁵ClNO₃, 452.1992), 42 amu (C₂H₂O) less than compound 1. Its ¹H NMR spectrum revealed nearly all identical structural features to those found in compound 1, except the absence of an acetyl signal in the ¹H NMR spectrum (Table 1) and thus the structure of compound 2 could be readily determined as shown in Figure 1 which was supported by 2D NMR spectra.

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Compound 3 was established the molecular formula as C₃₁H₃₃ClN₂O₄ by HRESIMS analysis (m/z 533.2203, calcd. for C₃₁H₃₄³⁵ClN₂O₄, 533.2207) with 16 degrees of unsaturation. The ¹H NMR spectroscopic data of compound 3 were well comparable to those of 1. Considering the molecular formula, an indole ring might substitute 2'-OH of isochromophilone Ⅵ in the compound 3. The HMBC (Figure 2) correlations from H-10' to C-4' and C-9', from H-5' to C-7' and C-9', from H-8' to C-4' and C-6' confirmed the presence of indole ring.

The 7R-configurations of isochromophilones Ⅹ–Ⅻ (1–3) were determined by circular-dichroism (CD) spectroscopy. The observed positive Cotton effect at the wavelength (370– 390 nm) was well comparable to that of 7R-5-chloro-7, 8-dihydro-3-(3-hydroxypropyl)-7-methyl-6, 8-dioxo-6H-2-benzo-pyran-7-yl ester.^{13, 14} Furthermore, the 14S-configurations of 1–3 were determined by comparing their NMR data of the side chain moieties with those of similar azaphilone compounds, isochromophilone Ia, IIa and rotiorinol A.^{15, 16}

Biosynthetically, azaphilone was derived from the polyketide pathway, ^{3, 17} whose oxygen atom could be further substituted by primary amines or ammonia into N-containing compounds rapidly.¹ For example, sclerotiorin could be converted to sclerotioramine (4) with ammonia.² Thus, compounds 1 and 3 were deduced as consequences of sclerotiorin reacting with phenylethanamine and tryptamine, the decarboxylated product of phenylalanine and tryptophan, respectively (Scheme 1). The further deacetylation of compound 1 gave compound 2. Also, we can deduce that compounds 1–3 possessed the same configurations as that of sclerotiorin (Figure 1) by their biosynthetic view.

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All compounds were evaluated for their cytotoxicities against MCF-7, SGC-7901, SW1116, A549 and A375 cell lines by MTT method. Only, isochromophilone Ⅹ (1) showed moderate cytotoxic activities with its IC₅₀ values of 14.90, 16.84, 24.15, 26.93 and 35.75 μM against MCF-7, SGC-7901, SW1116, A549 and A375 cell lines (Table 2), respectively, while the other compounds exhibited no activities with IC₅₀ values greater than 50 μM.

Experimental Section

General Experimental Procedures. HRESIMS spectra were recorded on an Agilent 6210 TOF LC/MS. CD spectra were obtained on a JASCO J-810 spectrometer. IR spectra were obtained on a Nexus 870 FT-IR spectrometer. NMR spectra were recorded on Bruker AV-500, AV-300 or DRX500 NMR spectrometer. Optical rotations were recorded on a Rudolph Autopol Ⅲ automatic polarimeter. UV spectra were recorded on a Hitachi U-3000 spectrophotometer. Silica gel (200–300 mesh; Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 gel (Pharmacia Biotech, Sweden) were used for column chromatography (CC). HPLC was performed with a Hitachi L-7110 pump, and UV detector L-7400 equipped with an Apollo C18 column (5 μm, 250 mm × 4.6 mm; Alltech Associates, Inc. Chicago, IL, USA).

The Fungal Material and Cultivation Conditions. The strain IFB-3lp-10 was isolated from the healthy leaves of Rhizophora stylosa collected in August 2010 from the mangrove forest of Hainan Province of China. The strain was identified as Diaporthe sp. by comparing the morphological character and 18S rDNA sequence with that of a standard record. A voucher specimen has been deposited in the Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University. After growing on PDA medium at 28 ℃ for 5 d, the fungus Diaporthe sp. IFB-3lp-10 was inoculated into Erlenmeyer flasks (1000 mL) containing 400 mL of ME liquid medium. After incubation for 4 d at 28 ℃ on a rotary shaker at 150 rpm, 20 mL of culture liquid was transferred as the seed into 250 mL flasks, each preloaded with the evenly mingled medium (7.5 g of grain, 7.5 g of bran, 0.5 g of yeast extract, 0.1 g of sodium tartrate, 0.01 g of FeSO₄·7H₂O, 0.1 g of sodium glutamate and 30 ml of H₂O). The fungus then grew for 30 d at 28 ℃ with the relative humidity in the range 60–70%.

Extraction and Isolation. The air-dried samples (30 kg) were extracted with 95% EtOH and the organic solvent was evaporated to dryness under a vacuum to afford a crude extract (1.2 kg) which gave six fractions (Fr.1, 21g; Fr.2, 75 g; Fr.3, 56 g; Fr.4, 25 g; Fr.5, 36 g; Fr.6, 158 g) upon column chromatography (10 × 120 cm) on silica gel (6000 g, 200–300 mesh) eluted with a gradient of CH₂Cl₂/MeOH (v/v 100:0, 100:1, 100:2, 100:4, 100:8, 100:20, 0:100, each 20L) based on TLC monitoring. Purification of the second subfraction (CH₂Cl₂/MeOH 100:1) by using column chromatography (petroleum ether/EtOAc 1:1) and Sephadex LH-20 (100% MeOH) followed by HLPC (90% MeOH) gave 1 (15.0 mg) and 2 (2.5 mg). Purification of the third subfraction (CH₂Cl₂/MeOH 100:2) in the same way mentioned above gave 3 (3.6 mg). The fourth fraction was subjected to reversed phase ODS column (4 cm × 40 cm) with a gradient of MeOH/H₂O (v/v 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, each 3 L) to give nine subfractions, respectively. Purification of the fifth subfraction (MeOH/H₂O 60:40) by using column chromatography (petroleum ether/EtOAc 1:2) and Sephadex LH-20 (100 % MeOH) gave 4 (2.8 g) and 5 (64 mg).

Isochromophilones X (1): red needles. [α]_D²⁰ = +1116.4 (c = 0.028, MeOH). UV/Vis (MeOH): λ_max/nm (log ε) = 201 (4.5), 227 (4.4), 373 (4.5). CD (c 2.8 × 10^-4 g ml^-1, MeOH) λ_max/nm (Δε): 229.5 (+0.4), 246 (+6.2), 304 (−8.9), 381.5 (+7.9), 429 (+1.2). IR (KBr): ν_max = 3490.4, 2960.7, 2924.7, 1701.8, 1594.6, 1501.6, 1262.5, 1220.3, 1144.3, 1081.7, 1032.0, 801.9 cm^-1. HRESIMS: m/z 494.2080 ([M + H]⁺, calcd. for C₂₉H₃₃³⁵ClNO₄, 494.2098). For ¹H and ¹³C NMR spectroscopic data, see Table 1.

Isochromophilones Ⅺ (2): red needles. [α]_D²⁰ = +596.0 (c = 0.08, MeOH). UV/ Vis (MeOH): λ_max/nm (log ε) = 206 (4.5), 228 (4.5), 350 (4.5). CD (c 8.0 × 10^-4 g ml^-1, MeOH) λ_max/nm (Δε): 209 (+5.6), 236 (+0.6), 249 (+1.4), 311 (−4.3), 375 (+3.6), 414.5 (+0.5). IR (KBr): ν_max = 3419.2, 2962.5, 1588.8, 1494.7, 1261.5, 1096.2, 1033.4, 801.5 cm^-1. HRESIMS: m/z 452.1998 ([M + H]⁺, calcd. for C₂₇H₃₁³⁵ClNO₃, 452.1992). For ¹H and ¹³C NMR spectroscopic data, see Table 1.

Isochromophilones Ⅻ (3): red needles. [α]_D²⁰ = +936.0 (c = 0.025, MeOH). UV/ Vis (MeOH): λ_max/nm (log ε) = 201 (4.6), 221 (4.7), 375 (4.3). CD (c 2.5 × 10^-4 g ml^-1, MeOH) λ_max/nm (Δε): 229.5 (−1.6), 246.5 (+2.4), 304.5 (−4.5), 379 (+3.5), 419.5 (+0.3). IR (KBr): ν_max = 3440.3, 3237.5, 2958.9, 1736.0, 1574.3, 1486.1, 1219.3, 1145.0, 758.9 cm^-1. HRESIMS: m/z 533.2203 ([M + H]⁺, calcd. for C₃₁H₃₄³⁵ClN₂O₄, 533.2207). For ¹H and ¹³C NMR spectroscopic data, see Table 1.

Cytotoxicity Assay. The cytotoxic effects of 1–5 on the viability of the SGC-7901, SW1116, MCF-7, A549 and A375 cell lines were assayed with the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] colorimetric method. The data represent the mean of three experiments performed in triplicate and are expressed as means ± SD.

Notes

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-012-0023-2 and is accessible for authorized users.

Acknowledgments

This work was co-financed by National Natural Science Foundation of China (81121062, 21132004, 90813036, and 81172948), MOE (NCET-10-0477), and Jiangsu Provincial Government (BK2009010).