Two new α-pyrone-containing polyketides isolated from the fungus Aspergillus aureoterreus

Abstract Two undescribed α-pyrone-containing polyketide derivatives designated aurovertins V (1) and W (2), and a known analogue (3), were isolated from the fungus Aspergillus aureoterreus. Their structures including the absolute configuration were elucidated on the basis of extensive spectroscopic methods and theoretical ECD calculation. Compound 1 is the first example of aurovertins with a 7R configuration, whereas 2 comprises a S configuration for C-6 and a Z geometry of the double bond Δ8. Both 1 and 2 showed no cytotoxicity against human cancer cell lines HL-60, SU-DHL-2 and U266) at the concentration of 20.0 μM. Graphical Abstract


Introduction
Aurovertins is a type of a-pyrone-containing polyketides, where a 2,6-dioxabicyclo[3.2.1]octane (DBO) skeleton constructed with the a-pyrone scaffold via a triene unsaturated alkyl linker. Until now, over 40 aurovertins and structurally analogues have been isolated from a number of fungal species, which shows a wide range of bioactivities such as antitumor, antiviral, and antibacterial activities (Mulheirn et al. 1974;Park et al. 2007;Azumi et al. 2008;Adachi et al. 2015;Wang et al. 2016;Zhao et al. 2016;Li et al. 2018;Zhu et al. 2020). Among them, aurovertins B, D, and M exhibited potential antitumor activity against human triple-negative breast cancer cell line (MDA-MB-231) with IC 50 values ranging from 0.08 to 0.25 lM reported by Zhao and colleagues (Zhao et al. 2016). Additionally, one of related compounds namely verrucosidin was isolated from Penicillium verrucosum var. cyclopium and showed significant inhibition of glucose-deprived HT-29 human colon carcinoma cells with a IC 50 value at 50 nM (Park et al. 2007). Comparing to aurovertins, their analogues comprised several cyclic ring systems rather than a DBO core which indicated more possibilities of structural diversity and activities.
As a continuous search for structurally novel and bioactive compounds from fungi, two new a-pyrone-containing polyketides namely aurovertins V (1) and W (2) were isolated from culture broth of Aspergillus aureoterreus GDMCC 3.619 (¼ CBS 503.56) together with a known derivative ( Figure 1). Both new structures and their absolute configurations were established by spectroscopic methods and theoretical electronic circular dichroism (ECD) calcualtion. Up to the best of our knowledge, compound 1 is the first example of aurovertins with a 7R configuration, whereas 2 comprises a S configuration for C-6 and a Z geometry of the double bond D 8 . Herein, the isolation, structural elucidation, and biological evaluation were reported.
Hence, we subsequently deduced the relative configuration from the ROESY spectrum. Key ROESY correlations between H-4 and Me-19/Me-20/Me-21, H-6 and Me-21 revealed that H-4, H-6, Me-19, Me-20, and Me-21 were on the same face of the pyran ring of DBO core as shown in Figure S2, whereas H-2 and 4-OH were co-facial deduced from a ROESY correlation of H-2/4-OH. In addition, three double bonds of the unsaturated alkyl chain were E geometries by three big coupling constants. Here, theoretical ECD calculation using TDDFT method at B3LYP-D3(BJ)/6-31G Ã level was applied to determine the absolute configuration of 1. As a result, the absolute configuration of 1 was demonstrated to be 2 R,3R,4S,5S,6S,7R by comparing of experimental and calculated ECD curves ( Figure S3).
Aurovertin W (2) was also obtained as yellow amorphous powder and the molecular formula was determined to be C 23 H 30 O 7 by HRESIMS with a positive ion peak at m/z 441.1883 [M þ Na] þ (calcd. for C 23 H 30 O 7 Na þ , 441.1884). The 1 D (Table S1), HSQC, COSY, and HMBC NMR ( Figure S1) spectra indicated a roughly same planar structure with those of compounds 1 and 3. One of obvious differences was a Z geometry of the double bond D 8 deduced from a small coupling constant at 6.3 Hz. Until now, three heterologous produced aurovertins were reported with a Z geometry for double bonds D 12 in iso-aurovertins C, E, and I . Analyses of ROESY spectrum showed a similar relative stereochemistry between 2 and 3 excepting the chiral center C-6. Cross peaks between H-6/Me-1/Me-21 in the ROESY spectrum of 2 suggested H-6/Me-1/Me-21 were in a co-facial position while H-6 was on the opposite side in 3 ( Figure S2). Subsequently, ECD calculation was used to conclude the absolute configuration. As shown in Figure S3, a calculated ECD curve of 2 R,3R,4S,5S,6R,7S-2 was well matched with the experimental one. Therefore, the structure of compound 2 was ambiguously determined.
The known compound was identified to be aurovertin U (3) by comparison of their NMR and HRESIMS data with the literature (Zhao et al. 2016). The cytotoxicity evaluation of 1-3 against human cancer cell lines (HL-60, SU-DHL-2 and U266) showed that only 3 exhibited a weak inhibition of 33.5% to HL-60 cells at the concentration of 20.0 lM.

General experimental procedures
Optical rotations were determined with a Rudolph Autopol IV automatic polarimeter. The UV, IR, and ECD spectra were obtained on a PerkinElmer LAMBDA 35 UV/Vis spectrophotometer, a Bruker Vertex 70 instrument, and a JASCO-810 ECD spectrometer, respectively. NMR spectra were recorded on a Bruker Ascend TM 600 MHz (d in ppm; Mult. J in Hz) spectrometer, with 1 H and 13 C NMR chemical shifts referenced to the solvent or solvent impurity peaks for DMSO-d 6 (d H 2.50 and d C 39.52). HRESIMS data were acquired using an electrospray ionisation (ESI) with a Thermo Scientific Q Exactive Focus spectrometer. Semi-preparative HPLC utilised a SHIMADZU Prominence LC-20AT quaternary system with a UV-VIS detector, using a reverse-phased YMC C18 column (5 lm, 10 Â 250 mm). Column chromatography (CC) was performed using silica gel (Qingdao Marine Chemical Inc., China), ODS (50 lm, YMC, Japan), respectively. Thin-layer chromatography (TLC) was performed with silica gel 60 F 254 (Yantai Chemical Industry Research Institute).

Fungus materials
The fungus Aspergillus aureoterreus CBS 503.65 was bought from Centraalbureau voor Schimmelcultures (CBS) and stored in Guangdong Microbial Culture Collection Center (GDMCC) of China with a code number GDMCC 3.619. ITS-rDNA sequence amplified by conserved primer ITS1 and ITS4 shows 100% identity with A. aureoterrus CBS 503.65 (¼NRRL 1923) by NCBI BLAST.

Extraction and isolation
The fungal strain was maintained on potato dextrose agar (PDA) medium at 28 C for 5 days used as the seed culture. The fermentation was carried out in 50 L liquid medium (PDB, 5 g peptone, 10 g mannitol, 2.5 g yeast extract, and 500 mL distilled water per 1 L Erlenmeyer flask) under shaken (160 rpm) for 16 days at 28 C. After incubation, the culture broth was filtrated and extracted with ethyl acetate (EtOAc) at room temperature to yield 38.0 g of residue. The EtOAc extract was subjected to a flash column chromatography (CC) over silica gel with a gradient elution using petroleum ether/EtOAc (50:1 to EtOAc neat, v/v) and EtOAc/MeOH gradient (10:1 to MeOH, v/v) to afford 8 fractions (Fr.1-Fr.8).

Computational method
In general, conformational analyses were carried out via random searching in the Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 5 kcal/mol. The results showed five lowest energy conformer for both compounds. Subsequently, geometry optimisations and frequency analyses were implemented at the B3LYP-D3(BJ)/6-31G Ã level in PCM methanol using ORCA5.0.1 (Neese 2012). All conformers used for property calculations in this work were characterised to be stable point on potential energy surface (PES) with no imaginary frequencies. The excitation energies, oscillator strengths, and rotational strengths (velocity) of the first 60 excited states were calculated using the TD-DFT methodology at the PBE0/def2-TZVP level in PCM methanol using ORCA5.0.1. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, sigma ¼ 0.30 for all) (Stephens and Harada 2010). Gibbs free energies for conformers were determined by using thermal correction at B3LYP-D3(BJ)/6-31G Ã level and electronic energies evaluated at the wB97M-V/def2-TZVP level in PCM methanol using ORCA5.0.1. To get the final spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (DG).

Cytotoxicity assay
HL-60 (Human promyelocytic leukemia cells), SU-DHL-2 (Human diffuse tissue lymphoma cells), and U266 (human multiple myeloma cells) tumor cells were purchased from the National Collection of Authenticated Cell Cultures of China. The cytotoxicity was determined by the MTS method as reported in our previous study (Zang et al. 2020). Colorimetric assays were performed to evaluate the activity of each compound.

Conclusions
In summary, two new a-pyrone-containing polyketide derivatives designated aurovertins V (1) and W (2) along with a known analogue were discovered from the culture broth of Aspergillus aureoterreus. Their structures including absolute configurations were established using comprehensive spectroscopic data and ECD calculations. Among them, compound 1 is the first example of aurovertins with a 7 R configuration, where 2 possesses a unique S configuration for C-6. The discovery of new compounds enriches the diversity of aurovertins which should be interested to chemists.