Isolation and structure elucidation of a new compound from the fungus Aspergillus flavipes PJ03-11

Abstract A new diphenyl ether 3-methylpentyl-2, 4-dichloroasterrate (2), along with a known diphenyl ether butyl 2, 4-dichloroasterrate (1) were isolated from the metabolites of a wetland fungus Aspergillus flavipes. PJ03-11. The structures of 1 and 2 were determined by extensive NMR and HR–ESI–MS experiments. Compounds 1 and 2 showed weak cytotoxic activity, but both of them showed no antimicrobial activity.

Diphenyl ether derivatives are kind of common fungi secondary metabolites. In the course of our ongoing studies of new bioactive natural products from the wetland fungus Aspergillus flavipes PJ03-11 Zhang Feng, Zhao et al. 2016), another new diphenyl ether 3-methylpentyl-2,4-dichloroasterrate (2), together with a known compound butyl 2,4-dichloroasterrate (1) (Lin et al. 2009) were obtained (Figure 1). Details of the isolation and structural elucidation of the compounds are presented here.

General experimental procedures
IR spectra were measured on a Bruker IFS-55 infrared spectrophotometer (Bruker Co., Zurich, Switzerland) in KBr discs. The NMR spectral data were recorded on Bruker AV-600 (400 MHz for 1 H NMR and 100 MHz for 13 C NMR) with TMS (tetramethylsilane) as the internal standard (Bruker Co.). The HR-EI-MS data were obtained on the Microssmass AutoSpec-UltimaE TOF mass spectrophotometer (Bruker Co.). Chromatography was carried out on silica gel (

Cytotoxicity assay
Cytotoxic activities of the isolates were evaluated by the MTT assay against the human leukaemia cell lines (HL-60), the human colon cell lines (HCT-116), human colon cancer cells (HT-29) and prostate cancer cell lines (PC-3). The cell lines were purchased from America Type Culture Collection, ATCC (Rockville, MD, USA) and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, New York, NY, USA) with 10% fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 1 mmol/L ʟ-glutamine, and 10% (v/v) heat-inactivated FBS in a humidified atmosphere of 5% CO 2 at 37.0°C. Appropriate dilutions of the test samples were added to the cultures. The growth inhibition was calculated comparing with control cells. 5-fluorouracil was used as a positive control.

Antimicrobial bioassay
The MIC values for all compounds were determined by the dilution method. For sample preparation, each of the test compounds was dissolved in DMSO and then diluted with sterile broth to a concentration of 500 μg/mL. Further dilutions of the compounds in the test medium were prepared at the required quantities of 250, 125, etc. down to 3.9 μg/mL. Chloramphenicol and fluconazole were used as positive controls for bacteria and fungus, respectively. The in vitro antimicrobial activity of the compounds was tested by tube-dilution technique using individually pack-aged, flat bottomed, 96-well microtitre plates (NCCLS. 2000). Bacterial strains were maintained on LB medium for 48 h at 37°C and fungal strains were on PDA medium for 48 h at 28°C. The cell density for bacteria was 2~4 × 107 CFU/mL and 2~4 × 105 CFU/mL for fungus. A serial dilution of compounds were performed in the microplates and incubated for 12 h. The last tube with no growth of micro-organism was recorded to represent the MIC value expressed in μg/mL.

Conclusions
In conclusion, a new diphenyl ether 3-methylpentyl-2, 4-dichloroasterrate (2), along with a known diphenyl ether butyl 2,4-dichloroasterrate (1) were isolated from the metabolites of a wetland fungus Aspergillus flavipes PJ03-11. Compounds 1 and 2 showed weak in vitro cytotoxic activity against both the human leukaemia cell line HL-60 and prostate cancer cell line PC-3. Compound 1 showed weak in vitro cytotoxic activity against the human colon cell lines HCT-116. Compound 2 showed weak in vitro cytotoxic activity against human colon cancer cells HT-29. Both of them showed no antimicrobial activity.