Pyrenocines are better known as phytotoxic metabolites been found in Alternaria, Curvularia, Penicillium and Phomopsis strains. The class of compounds showed several biological activities, reportedly being phytotoxic, antifungal, cytotoxic, antitrypanosomal and antimalarial.1, 2, 3 Fungi of the genus Colletotrichum, a class of the widely distributed endophytic fungi, were reported as prolific producers of bioactive metabolites.4, 5, 6, 7 However, there are few reports on pyrenocines found in genus Colletotrichum. During our ongoing chemical investigations of endophytic fungi as sources of new cytotoxic natural products, the organic solvent extract of solid substrate fermentation culture of a Colletotrichum sp. HCCB03289 strain displayed cytotoxicity against cancer cell lines. Bioassay-guided fractionation of the extract led to the isolation of two novel pyrenocines N–O (1–2), and five known pyrenocines macommelin-9-acetate (3),8 pyrenocine A (4),9 pyrenocine B (5),9 pyrenocine E (6)10 and novae-zelandin A (7).11 Details of the isolation, structure elucidation and the cytotoxic activities of these compounds are presented here.

The culture of Colletotrichum sp. HCCB03289 was isolated from traditional Chinese medicinal plant Ludwigia prostrata Roxb specimen collected from Tianmu mountain, Zhejiang Province of China, in July 2008. The isolation and identification methods of endophytic fungal strain HCCB03289 were carried out as reported.12 The ITS (internal transcribed spacer) sequence data were submitted to GenBank with accession no. KF594420. Its ITS sequence was similar to that of Colletotrichum gloeosporioides strain HGUP0020 (maximal identities: 100%), but due to the lack of a sexual structure in the culture, the endophytic fungus was then identified at the genus level only as Colletotrichum sp. The fungal strain was cultured on slants of potato dextrose agar at 28 °C for 5 days. Then, 5.0 ml of spore suspension (spore/cell 1 × 106 ml−1) was inoculated into 500 ml Erlenmeyer flask ( × 100) each with 80 g of rice medium at 28 °C for 30 d. The fermented rice substrate was freeze-dried and extracted with ethyl acetate (10 litres) subsequently. The organic solvent was evaporated under vacuum to obtain a crude extract (20.0 g), which was fractionated by silica gel column chromatography (2.6 × 40 cm) using petroleum ether/ethyl acetate (7:3, 6:4, 3:7, 1:9) gradient elution to afford four fractions, Frs. 1–4. The Fr. 1 (1.0 g) was further separated by Sephadex LH-20 (Pharmacia, Uppsala, Sweden) eluted with CHCl3/CH3OH (10:1) to yield a subfraction of 200 mg, which was then purified by semi-prep. RP-HPLC (YMC-Pack RP-C18 column (YMC, Kyoto, Japan), 10 × 250 mm, 65% CH3OH in H2O for 40 min, 2.5 ml min−1) to yield novae-zelandin A (7, 6.5 mg; tR 28.3 min). The Fr. 2 (1.5 g) was further separated by Sephadex LH-20 eluted with CHCl3/CH3OH (5:1) to yield a subfraction of 150 mg. Purification of the subfraction by semi-prep. RP-HPLC (10 × 250 mm, 45% CH3OH in H2O for 30 min, 2.5 ml min−1) afforded pyrenocine E (6, 11.6 mg; tR 23.5 min). The Fr. 3 (1.2 g) was further separated by semi-prep. RP-HPLC (YMC-Pack RP-C18, 20 × 250 mm, 50% CH3OH in H2O for 15 min, gradient to 100% in 30 min, 7 ml min−1) to afford two subfractions, Frs. 2.1–2.2. The Fr. 2.1 (12.3–13.2 min, 90 mg) was then purified by semi-prep. RP-HPLC (10 × 250 mm, 35% MeOH in H2O for 30 min, 2 ml min−1) to yield pyrenocine B (5, 8.7 mg; tR 14.5 min). The Fr. 2.2 (15.2–16.5 min, 0.12 g) was then purified by semi-prep. RP-HPLC (10 × 250 mm, 35% MeOH in H2O for 40 min, 2 ml min−1) to yield pyrenocine A (4, 15.0 mg; tR 31.5 min) and macommelin-9-acetate (3, 10.6 mg; tR 36.5 min). The Fr. 4 (200 mg) was purified by semi-prep. RP-HPLC (10 × 250, 20% MeOH in H2O for 40 min, 2 ml min−1) to afford pyrenocine N (1, 12.5 mg; tR 32.1 min) and pyrenocine O (2, 23.6 mg; tR 30.6 min). UV data were recorded on a Shimadzu Biospec-1601 spectrometer (Shimadzu, Kyoto, Japan). IR data were recorded using a Nicolet 6700 FT-IR spectrometer (Thermo-Fisher, Madison, WI, USA) with KBr disks. Optical rotations were measured by JASCO P-2000 spectropolarimeter (Jasco, Tokyo, Japan). 1H and 13C NMR data were acquired with a Bruker Avance-500 (Bruker, Fällanden, Zurich, Switzerland) and Bruker Avance-400 spectrometer using solvent signals (CDCl3; δH 7.26/δC 77.6, CD3OD; δH 3.31/δC 49.0) as references. HRESIMS data were acquired using a Waters QTOFMS Premier spectrometer (Waters, Milford, MA, USA).

Pyrenocine N (1) was obtained as yellow oil. The molecular formula C10H12O4 was determined by positive HR-ESI-MS at m/z 197.0808 [M+H]+requiring five degrees of instaurations. The IR spectrum suggested the presence of hydroxyl group (3405 cm−1) and carbonyl group (1700 cm−1). The 1H, 13C and HSQC spectra of 1 revealed the presence of two methyl groups, one methylene unit, two oxygenated methines, four olefinic carbons and one carboxyl carbon. These data (Table 1 and Supplementary Fig. S1–S5) accounted for all 1H and 13C resonances except one exchangeable proton and suggested 1 contained two rings. The presence of the C-4 oxygenated pyrone moiety was evident from δH 5.36 (s, H-5) in the 1H NMR spectrum, and δC 176.0 (C-4), 168.5 (C-6), 157.3 (C-2), 113.4 (C-3) and 85.0 (C-5) in the 13C NMR spectrum.13 Analysis of the 1H, 1H-COSY NMR data led to the identification of one isolated 1H spin-system corresponding to the C-7 to C-10 subunit of structure 1. HMBCs (Figure 1) of H2-7 to C-2, C-3 and C-4 and of H-8 with C-4 permitted completion of the dihydrofuran subunit. Correlations from H3-11 to C-2 and C-3 indicated the connection of C-11 to C-2. The remaining hydroxyl could be located at C-9 corresponding the 13C-NMR chemical shift 68.6. On the basis of these data, the gross structure of 1 was characterized (Figure 2).

Table 1 1H(500MHz, CDCl3) and 13C NMR (100MHz, CD3OD) spectra data of 1 and 2 (δ in p.p.m., J in Hz)
Full size table
Figure 1
figure 1

1H–1H COSY and key HMBCs of 1. A full color version of this figure is available at The Journal of Antibiotics journal online.

Full size image
Figure 2
figure 2

Structures of compounds of 1–7.

Full size image

Pyrenocine O (2) was obtained as yellow oil, which assigned the same molecular formula C10H12O4 as pyrenocine N (1) by positive HR-ESI-MS at m/z 197.0812 [M+H]+. Analysis of the 1H and 13C NMR data (Table 1 and Supplementary Fig. S9–S13) for 2 revealed the presence of nearly identical structural features to those found in 1, except that the chemical shifts for the two oxymethines C-8 (δH/δC 4.78/92.4 in 1; 4.75/92.3 in 2) and C-9 (δH/δC 4.12/68.6 in 1; 3.86–3.90/69.6 in 2), as well as the 1H–1H coupling constant observed between H-8 and H-9 were different. These data implied that 2 was a stereoisomer of 1, and this conclusion was supported by analysis of its COSY and HMBC data.

The relative configuration of compounds 1 and 2 was assigned by NOED data (Supplementary Fig. S6). Upon irradiation of H3-10, enhancement was observed for H-8 in the NOE difference spectrum of 1, suggesting a cis relationship between H3-10 and H-8, whereas enhancement was not observed for H-8 in the spectrum of 2, indicating a trans relationship between H3-10 and H-8.

The absolute configuration of pyrenocine N (1) was assigned by application of the modified Mosher method.14, 15 Treatment of 1 with (S)-MTPA Cl and (R)-MTPA Cl afforded the R-MTPA ester (1a) and S-MTPA ester (1b), respectively. The difference in chemical shift values (Δδ=δS–δR) for the diastereomeric esters 1b and 1a were calculated in order to assign the absolute configuration at C-9 (Figure 3 and Supplementary Fig. S7,S8).

Figure 3
figure 3

ΔδS–R values of MTPA esters of 1.

Full size image

Negative Δδ values were observed for H-10 (−0.08) and H-8 (−0.03), while positive Δδ values were observed for Hα-7 (+0.08), Hβ-7 (+0.07), H-5(+0.02) and H-11 (+0.01), indicating the R absolute configuration at C-9 (Figure 2). Accordingly the configurations at C-8 and C-9 were assigned as 8S and 9R for 1. Therefore, the absolute configuration of 2 was deduced as 8S and 9S.

Pyrenocine N (1): Yellow oils. (c 0.7, CH3OH). UV (CH3OH): 238 (log ɛ 2.43), 285 (log ɛ 3.66). IR (neat) νmax 3405 (br), 2975, 2925, 1700, 1656, 1602, 1440, 1272, 1222 cm–1. 1H- and 13C-NMR: see Table 1. HRESI-MS (positive) m/z: 197.0808([M+H]+, C10H13O; calcd 197.0814).

Pyrenocine O (2): Yellow oils. (c 0.20, CH3OH). UV (CH3OH): 238 (log ɛ 2.17), 285 (log ɛ 3.40). IR (neat) νmax 3423 (br), 2977, 2929, 1706, 1602, 1434, 1216 cm–1. 1H- and 13C-NMR: see Table 1. HRESI-MS (positive) m/z: 197.0812([M+H]+, C10H13O; calcd 197.0814).

Compounds 1–7 were evaluated for cytotoxic activities against three human cancer cell lines: A549, MDA-MB-231 and PANC-1 in vitro using the MTT method (Table 2). Compound 4 showed most potent antitumor activities with CC50 values less than 4.0 μg ml−1 against cancer cell lines, which was stronger than that of 5-fluorouracil (the positive control). Compounds 5–6 displayed significant cytotoxicity, whereas compounds 1–3 have no cytotoxic activities. Based on the analysis of cytotoxicity potencies and the structural characteristics of 4–6 and 1–3 or 7, it was found that 3-ketone group in branched chain (4–6) would significantly increase the cytotoxic activities. Moreover, α-, β-unsaturated ketone in branched chain of compound 4 could further strengthen cytotoxicity than that of 5 or 6.

Table 2 Cytotoxic activities of compounds 1–7
Full size table