Metabolites Produced by the Endophytic Fungus Aspergillus fumigatus from the Stem of Erythrophloeum fordii Oliv.

A new diketopiperazine alkaloid named spirotryprostatin K (1), and five known alkaloids, spiro[5H,10H-dipyrrolo[1,2-a:1′,2′-d]pyrazine-2(3H),2′-[2H]-indole]-3′,5,10(1′H)trione (2), 6-methoxyspirotryprostatin B (3), pseurotin A (4), N-β-acetyltryptamine (5), and lumichrome (6) were isolated from the endophytic fungus Aspergillus fumigatus. The structure and the absolute configuration of spirotryprostatin K were established by extensive spectroscopic analyses, acid hydrolysis and ECD calculations. Pseurotin A exhibited indirect anti-inflammatory activity by suppressing the lipopolysaccharide-induced proinflammatory factors in BV2 microglial cells, with an IC50 of 5.20 µM.


Introduction
Interest in endophytic fungi as sources of novel bioactive compounds is increasing because bioactive natural products from endophytic microbes have shown enormous potential as sources of new medicinal OPEN ACCESS and agricultural products [1,2]. Considering that the relationship between endophytic fungi and host plants may range from latent phytopathogenesis to mutualistic symbiosis [1], endophytic fungi residing in poisonous plants may produce structurally diverse secondary metabolites as a consequence of their biological and biochemical evolution. We therefore initiated a research program focusing exclusively on the discovery of secondary metabolites from endophytic fungi associated with poisonous plants.

Results and Discussion
Compound 1 was obtained as a yellow amorphous powder and was assigned a molecular formula of C21H25N3O4 based on HRESIMS (m/z 406.1751 [M + Na] + ; calcd 406.1737) and NMR data ( Table 1). The UV spectrum showed maxima ascribable to a substituted benzene ring (213 and 270 nm), and the IR spectrum showed the presence of amide carbonyl (1692 and 1636 cm −1 ) and hydroxyl (3212 cm −1 ) groups. The 1 H-NMR data (Table 1)  , and two methyl signals, δH 1.51 (3H, s, Me-21) and 1.56 (3H, s, Me-22), along with signals due to several methine and methylene groups. The 13 C-NMR data ( Table 1) revealed twenty-one carbon resonances, including three amide carbonyls at δC 184.0 (C-2), 170.6 (C-10), and 166.1 (C-13), one oxygen-bearing sp 2 carbon at δC 159.8. According to the above features, the NMR spectroscopic data of 1 were very similar to those of spirotryprostatin A [10], the major difference between 1 and spirotryprostatin A being that the methine carbon (δC 60.2, C-18) in spirotryprostatin A was replaced by a methylene carbon (δC 38.2, C-18) in 1. The chemical shift changes and analyses of the degrees of unsaturation of 1 and spirotryprostatin A showed that the bond between C-18 and N-14 in spirotryprostatin A was broken in 1, which was confirmed by the key HMBC correlations from H2-18 to C-2/C-20 and from H-14 to C-9/C-12/C-13 ( Figure 2). The other difference between 1 and spirotryprostatin A was that the methoxy (δC 55.5, δH 3.80) in spirotryprostatin A was replaced by a hydroxyl in 1.  The relative configurations of C-9 and C-12 were deduced from the NOESY and NOE spectra ( Figures S8 and S9 in Supporting Materials). NOESY correlation of H-9 with H-12 indicated that H-9 and H-12 were on the same face of ring C. A strong NOE was observed for H-12 after irradiation of H-9, which also indicated that H-9 and H-12 were on the same face of ring C. Marfey's method [10] was applied to assign the absolute configuration of the proline residue resulting from acid hydrolysis of 1. HPLC analysis of the 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) derivatives of the acid hydrolysate of 1 gave the same retention time as that prepared from sample of authentic L-proline (Figures S13-S15 in Supplementary Materials). Therefore, the proline residue in 1 was assigned the L-configuration. Considering the relative configurations established between C-9 and C-12 by NOESY and NOE data, the absolute configuration of C-9 was determined to be S. According to the above analyses, the possible absolute configurations of 1 were proposed to be (3S, 9S, 12S) (1a) or (3R, 9S, 12S) (1b). The calculated ECD spectrum of 1a displayed a CD curve similar to the experimental spectrum of 1 (Figure 3). Thus, the structure of 1 was determined as shown in Figure 1, and named spirotryprostatin K.  The isolated compounds 4 indirectly exhibited anti-inflammatory activity by suppressing lipopolysaccharide-induced NO production in mouse macrophages with IC50 values of 5.20 μM, Dexamethasone was used as the positive control, with IC50 = 2.5 × 10 −2 μM. The other isolated compounds 2-6 were inactive (IC50 > 10 μM) for the inhibition of NO production.

Fungal Material
The fungus Aspergillus fumigatus was separated from the stem of Erythrophloeum fordii Oliv.

Absolute Configuration of 1
An amino acid (L-proline or D-proline standard, 0.2 mg, Sigma, St. Louis, MO, USA) was dissolved in H2O (30 μL) and treated with 1% 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) in acetone (60 μL) and 6% Et3N in 30 μL of H2O at 40 °C for 1 h. After cooling to room temperature, the derivative was analyzed by reversed-phase HPLC with detection by UV absorption at 340 nm. The column [Agilent C18, 5 μm, 4.6 × 150 mm] was eluted with 35% CH3CN in H2O (0.1% trifluoroacetic acid) for 30 min. The standards gave the following retention times: 20.17 min for L-proline, and 23.90 min for D-proline. Compound 1 (1 mg) was hydrolyzed in 6 HCl (0.5 mL) and heated at 150 °C in a sealed vial for 1.5 h to yield the corresponding amino acids. The cooled reaction mixture was evaporated to dryness under reduced pressure, and HCl was removed from the residual acid hydrolysate by repeated evaporation from frozen H2O (1 mL). The amino acid mixture was then treated in the same manner as the standards above (1% FDAA and 6% Et3N). The mixture of FDAA derivatives was filtered, and the filtrate was diluted with H2O and analyzed by HPLC. The FDAA derivative of the amino acid liberated from 1 showed the peak at 20.14 min, matching the retention time of L-proline [11].

In Vitro Anti-Inflammatory Activity Assays
C57BL6/J mouse macrophages were cultured in 48-well plates in RPMI1640 medium at 37 °C for 24 h. Then, the cells were divided into four groups: the blank control (RPMI1640 medium only), the LPS control (1 μg/mL LPS in RPMI1640 medium), the experimental control (1 μg/mL LPS and the candidate compounds in RPMI1640 medium), and the positive control (10 −6 M dexamethasone in RPMI1640 medium). The cells were then incubated at 37 °C for an additional 24 h. From each well, a total of 100 μL of the supernatant was mixed with the same amount of Griess reagent, and the absorbance value was measured at 570 nm using a microplate reader. Sodium nitrite was used as the standard to calculate the NO2 − concentration [12,13].

Conclusions
A new diketopiperazine alkaloid, spirotryprostatin K (1), was isolated from the endophytic fungus Aspergillus fumigatus. Its structure and absolute configuration were determined by a combination of extensive spectroscopic methods, acid hydrolysis, and ECD calculations. A known alkaloid, pseurotin A (4) exhibited indirect anti-inflammatory activity by suppressing the lipopolysaccharide-induced proinflammatory factors in BV2 microglial cells, with an IC50 of 5.20 μM.