Two New Indolyl Diketopiperazines, Trypostatins C and D from Aspergillus penicilliodes Speg.

Twelve indolyl diketopiperazines 1–12 were isolated from the mycelia culture of Aspergillus penicilliodes Speg., a dominant microorganism from the post fermentation process of ripe Pu-er tea. Their structures were elucidated by extensive spectroscopic methods. Among them, trypostatins C (1) and D (2) featuring with a rare methyl vinyl ketone side chain at C-2 are new compounds, while 3 and 4 were obtained for the first time from nature source. The isolates 3–12 did not show obvious cytotoxicities against five human cancer cell lines at a concentration of 40 μM.

Aspergillus penicilliodes Speg. was isolated previously from the post-fermentation process of ripe Pu-er tea by our group [13]. In the research of the characteristic components responsible for health improvement of ripe Pu-er tea, the strain KIBA0502 of A. penicilliodes was studied chemically for its metabolites. This led to the identification of 12 indolyl diketopiperazines. Trypostatins C (1) and D (2) featuring with a rare methyl vinyl ketone side chain at C-2 are new compounds, while 3 and 4 were obtained for the first time from nature source. Their structures were determined by extensive spectroscopic analysis and by comparison with literature values. Most of the isolates were tested for their cytotoxicities against five human cancer cell lines.

Results and Discussion
The ethyl acetate extract of the mycelia culture of A. penicilliodes (strain KIBA0502) isolated previously from the fermentative stacks of Pu-er tea was applied to repeated column chromatography over silica gel and Sephadex LH-20, followed with semi-preparative HPLC, to afford 12 . From the well-defined coupling sequences and spatial relationships, three sub-structural fragments could be constructed for 1, consisting of one 2,3,6-trisubstituted indole, one 2,5-diketopiperazineand one methyl vinyl ketone moieties. These were subsequently reinforced by correlative interpretation of the 2D NMR spectra (Fig. 2), which allowed the unambiguous assignment of all the 1 H and 13 C NMR signals. The 1 H-1 H COSY correlations constructed obviously three partial structures of -C (4) H-C (5) H-, -C (8) H-C (9) H-NH-and -C (12) H-C (13) In the HMBC spectrum, correlation of the methoxy protons at d H 3.81 with C-6 (d C 159.7) indicated its location on C-6. The HMBC correlations of confirmed the presence of methyl vinyl ketone partial structure, which were determined to be connected to the indolyl C-2 by the HMBC correlations of H-19 (d H 6.09) with C-2 (d C 131.7) and H-18 (d H 6.76) with C-3 (d C 117.7). Comparison of the NMR data of 1 with those of tryprostatin A [14] manifested that 1 had a similar indolyl diketopiperazine skeleton, except for the presence of a methyl vinyl ketone side chain in 1, instead of an isoprenyl group in tryprostatin A. The relative configuration of 1 was established by ROESY experiment. In which, correlation of H-9 with H-12 indicated both protons located at the same  side. Based on comparison of its specific rotation with that of tryprostatin A [15], the absolute stereochemistry at C-9 and C-12 in 1 was assigned to be both as S configurations, which are the same to those of the other known 2,5-diketopiperazine-type compounds. On the basis of the above evidences, the structure of tryprostatin C (1) was deduced as shown in Fig. 1.
Compound 2 obtained as yellow amorphous powder, had a molecular formula of C 21 H 23 N 3 O 4 , as determined by the HRESIMS (m/z 404.1581 [M?Na] ? ), which was same to that of 1. The 1D NMR spectroscopic data were almost the same with those of 1, suggesting that 2 had the same indole diketopiperazine skeleton. The only different between 2 and 1 was the appearance of a trans-coupled double bond [(d H 7.48, 6.36, each 1H, d J = 16.0 Hz, H-18, H-19)] in 2, instead of the cis form in 1. The 2D NMR spectra further revealed that structure of 2 as shown in Fig. 1. The configuration of 2 was deduced to be the same as that of 1, according to the ROESY correlation of H-9 with H-12, and further confirmed by the specific rotation. Therefore, the absolute configuration of tryprostatin D (2) was established to be 9S,12S.
It is noted, at the room temperature, compound 2 changed rapidly to 1 in a methanol or chloroform solutions. The cis conformation of double bond in 1 is obviously more stable than the trans form in 2. This is opposite to the most cases that trans-coupled double bonds are more stable than the cis ones. It might be the formation of an intramolecular hydrogen bond between 1-NH and the C-20 ketone that caused the C-18/C-19 double bond to be cis form in 1.
The known compounds 3-12 were evaluated for their cytotoxic activities against human myeloid leukemia HL-60, hepatocellular carcinoma SMMC7721, lung cancer A-549, breast cancer MCF-7, and colon cancer SW480 cell lines. However, no compounds showed obvious activity at a concentration of 40 lM.

General Experimental Procedures
Optical rotations were measured with a P-1020 polarimeter (JASCO, Tokyo, Japan). UV spectra were recorded on a Shimadzu UV2401A ultraviolet-visible spectrophoto-meter. HREIMS spectra were run on a Waters Autospec Premier P776. NMR spectra were measured in CD 3 OD and recorded on a Bruker DRX-600 spectrometer, using TMS as an internal standard. Chemical shifts were reported in units of d (ppm) and coupling constants (J) were expressed in Hz. Column chromatography (CC) were carried out silica gel (200-300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China), Sephadex LH-20 (25-100 lm, GE Healthcare Bio-Science AB). An Agilent series 1260 (Agilent Technologies) were used for semi-preparative HPLC with an Agilent ZORBAX SB-C18 column (5 lm, 250 mm 9 9.4 mm). TLC was performed on pre-coated TLC plates (0.2-0.25 mm thickness, GF254 Silica gel, Qingdao Hailang Chemical Co. Ltd., Qingdao, China) with compounds visualized by spraying with anisaldehyde-sulfuric acid reagent and careful heating.

Fungal Material
Aspergillus penicilliodes Speg. was isolated previously by our group from the fermentative stacks of Pu-er tea produced at Puer specific tea factory in Puer city, Yunnan Province, China, and deposited in China general microbiological culture collection center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences [13]. A loop of spores from a colony growing on potato dextrose agars was inoculated into potato dextrose seed broth (20 g glucose, 200 g potato, 4 g peptone, 0.5 g MgSO 4 , 1.0 g KH 2 PO 4 , and 1000 mL distilled water) for 3 days in the dark at 28°C, 140 r/min. The seed culture (10 mL) was inoculated into 100 9 500 mL Erlenmeyer flasks, each containing 100 g of wheat immersed in waterand sterilized. Fermentation was conducted under stationary condition for 20 days in the dark at 28°C.

Extraction and Isolation
The mycelia culture was extracted three times with

Cytotoxicity Assay
Five human cancer cell lines, myeloid leukemia HL-60, hepatocellular carcinoma SMMC7721, lung cancer A-549 cells, breast cancer MCF-7, and colon cancer SW480, were used in the cytotoxic assay. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA).The cytotoxicassay was performed according to the MTS method in 96-well microplates. Briefly, adherent cells (100 lL) was seeded into each well of 96-well cell culture plates and allowed to adhere for 24 h before drug addition, while suspended cells were seeded just before drug addition, each tumor cell line was exposed to the test compound dissolved in DMSO in triplicates for 48 h at 37°C, with DDP and taxol (Sigma, USA) as positive controls. After the incubation, 20 lL MTS and 100 lL medium was added to each well after removal of 100 lL medium, and the incubation continued for 4 h at 37°C. The optical density was measured at 492 nm using a Multiskan FC plate reader (Thermo Scientific, USA).

Conclusion
In this study, 12 indole diketopiperazines 1-12 were identified for the first time from A. penicilliodes. Among them, 1 and 2 are new compounds, while 3 and 4 are new natural products. So far, the natural diketopiperazines with anti-cancer activities were mainly isolated from Aspergillus and Penicillium genera, with prenylated side chain as the most representative structural fragment, accounting for the majority of this class of compounds. This is the first time to have obtained indole diketo-piperazines featuring with a methyl vinyl ketone side chain at C-2.