An Alkaloid and a Steroid from the Endophytic Fungus Aspergillus fumigatus

Two new compounds, fumitremorgin 12-methoxy-13-[5'-hydroxy-2'-(1''-hydroxy-3''-methoxy-5''-methylbenzoyl)-3'-methoxy]benzoic acid methyl ester (fumitremorgin D, 1) and 4,8,10,14-tetramethyl-6-acetoxy-14-[16-acetoxy-19-(20,21-dimethyl)-18-ene]-phenanthrene-1-ene-3,7-dione (2) were isolated from the cultured endophytic isolated fungus Aspergillus fumigatus, together with fourteen known compounds. Their structures were elucidated by 1-D and 2-D NMR analyses. The cytotoxicity profile of the compound against the human hepatocellular carcinoma cell line HepG2 was evaluated by MTT antiproliferative assays.


Results and Discussion
Compound 1 was obtained as a yellow amorphous powder.The IR spectroscopic data indicated the presence of ether groups (1107 cm −1 and 1241 cm −1 ), amide groups (1631 cm −1 ), an amine group (3431 cm −1 ) and an ester group (1723 cm −1 ).Its molecular formula was determined as C40H41N3O11 by HR-ESIMS (m/z 740.2806 [M+H] + , C40H42N3O11 + calc.740.2814), requiring 22 sites of unsaturation for the whole molecule.All 40 carbons and 38 of 41 protons can be identified in the 13 C-and 1 H-NMR spectra of compound 1, and HSQC correlations suggest the presence of three exchangeable protons.Salient features of the molecule including seven methyl singlets, three sp 3 -hybridized methylenes, three sp 3 -hybridized and eight sp 2 -hybridized methines, one sp 3 -hybridized and fourteen sp 2 -hybridized quaternaries, one ketone (δ 199.9) and three ester or amide carbonyls were indicated by the NMR data.
By comparison with the literature data [11], fragment B in compound 1 was most likely an indole-diketopiperazine skeleton (Figure 1), which is indicated by the carbon combination, along with the presence of an amide group and amine group.This deduction was further confirmed by a correlative interpretation of its NOESY and HMBC spectrum (Table 1).Although no direct HMBC correlation between the methoxyl group (δ 3.62 (3H, s), δ 52.1) and C-12 (δ 83.1) was observed, the chemical shift of C-12 at δ 83.1 indicated that the methoxyl group was linked at C-12.
Structurally, this compound is the first example of a helvolic acid derivative possessing a phenanthrene skeleton.The cytotoxic activities of all compounds were evaluated by an MTT assay using the HepG2 cell line.Compounds 1 and 2 both showed weak cytotoxicity in this assay, with IC50 values of 47.5 μM and 139.9 μM, respectively.It was noteworthy that 12,13-dihydroxyfumitremorgin C (4), and verruculogen (5) showed cytotoxic activity against the HepG2 cell line with IC50 values of 4.5 μM and 9.8 μM, respectively.Despite the lack of a macrocyclic structure, 12,13-dihydroxyfumitremorgin C (4) showed improved activity over verruculogen (5), suggesting that the macrocyclic linking at 1-N and 3-C does not play a crucial role in the observed cytotoxicity.Meanwhile, compounds 1, 3 and 6 lacking C-12 and/or C-13 hydroxyls, showed IC50 values of 47.5, 156.5 and 44.9 μM, respectively.Based on above results, the simultaneous presence of hydroxyls at C-12 and C-13 showed an important structure-activity relationship (SAR) to the cytotoxic activity of indolediketopiperazine alkaloids against the HepG2 cell line.

Isolation and Cultivation of the Fungus
All plant tissues (mainly roots or rhizomes) of D. sinensis L. which were collected from Honghegu (Shanxi Province, China), were cleaned in running tap water and any visibly diseased or damaged material was eliminated.All tissues were surface disinfected with 75% ethanol for 1 min and thrice in sterile distilled water, then disinfected for 5 min in 0.1% mercuric chloride solution and thrice in sterile distilled water and step 1 was then repeated again.After surface sterilization and removal of epidermis the phloem was cut into 0.5-1 cm fragments and inoculated on potato dextrose agar medium (five pieces each) for 3-10 days at 28 °C.Individual colonies were transferred onto new potato dextrose agar medium for further analysis and maintenance.Based on 16s rDNA sequence analysis, strain Wrq12 was classified as A. fumigatus.The strain A. fumigatus Wrq12 is deposited in the China General Microbiological Culture Collection Center (CGMCC No. 3785).

Cytotoxic Activity Assays
Cytotoxicity was evaluated by the MTT method using HepG2 cell lines.The cell line was grown in DMEM supplemented with 10% FBS under a humidified atmosphere of 5% CO2 and 95% air at 37 °C.Then 100 μL of these cell suspensions at a density of 5 × 10 3 cell per well was plated in 96-well plates and incubated for 16-18 h under the above condition.Then the test compound solutions (in DMSO) were mixed with culture medium and cells were treated with them at gradient concentrations (12.75 μM, 25 μM, 50 μM, 100 μM, 200 μM).After further incubation under the same condition for 24 h, 10 μL of the MTT solution (5 mg/mL in DMEM medium) was added to each well and incubated for 4 h.The old medium containing MTT was then gently replaced by DMSO and standing 20 min to dissolve formazan crystals.Absorbance was then determined on an IMark (Bio-Rad, Hercules, CA, USA) plate reader at 490 nm.

Figure 1 .
Key HMBC and NOESY correlations for compound 1.