Two new quinazoline alkaloids produced by Aspergillus versicolor and their antimicrobial activities

Abstract Two new quinazoline alkaloids versicomides G–H (1 and 2), together with seven known compounds, were isolated from Aspergillus versicolor HYQZ-215 obtained from the sediment of Qarhan Salt Lake. Their structures were elucidated by NMR, HRESIMS, and quantum chemical ECD calculations data. The antimicrobial activities of these compounds were evaluated against seven agricultural pathogenic fungi and eight clinically drug-resistant bacteria.


Introduction
To survive in the extreme environments such as deep sea, polar, and hot regions, microorganisms have evolved unique strategies to adapt in these harsh conditions and biosynthesize novel bioactive metabolites, which are potentially useful for pharmaceutical, nutraceutical, and biotechnological applications [1,2].Qarhan Salt Lake, as the largest salt lake in China, is rich in inorganic salts and halophilic microorganisms [3].The extremely high salinity and drought ecological environment may promote fungi to produce unique metabolism and defense system in the process of natural evolution, which resulted in discovery of novel molecules with extensive biological activity [4,5].

Results and discussion
To understand the relative configuration, we acquired the nuclear Overhauser effect spectroscopy (NOESY) spectrum of 1.The observed NOESY correlation between 3-OH and H-15 indicated that 3-OH and H-14 were on the opposite side (Figure 2).Subsequently, the electronic circular dichroism (ECD) calculations of possible stereoisomers [(3R,14R,18S-1a), (3 R,14R,18R-1b), (3S,14S,18R-1c), and (3S,14S,18S-1d)] were performed.According to the result shown in Figure 3, the calculated ECD curves of 1a and 1b were in agreement with the experimental curve, suggested the absolute configuration of pyrazino ring was 3 R, 14 R. Referring to the stereochemistry of derivative 3, we speculated C-18 as S based on the common biosynthetic origin.
Compound 2 was isolated as yellow oil, with one more degree of unsaturation than 1 on the basis of HRESIMS data at m/z 340.1677 [M À H] À .Similar 1D NMR data (Table 1) indicated compounds 1 and 2 were analogues, excepted two additional sp 2      2).Thus, the planar structure of 2 was elucidated.The Z-configuration of the double bond D 3 (18) was determined by the NOESY correlation between 2-NH (d H 8.25) and H-20 (d H 2.31) (Figure 2).As shown in Figure 4, identical calculated ECD curve of 2a and experimental one assigned the configuration of C-14 as R, and named versicomide H.
Compounds 1À9 were evaluated the antimicrobial activities against eight clinically drug-resistant bacteria and seven agricultural pathogenic fungi.However, none of the evaluated compounds were active against the selected microbia (Tables S2 and S3).

Fungal material
The strain was isolated from the surface of sediment collected from Qarhan Salt Lake, Qinghai Province, China, in August 2018.The internal transcribed spacer (ITS) region of the rDNA sequence was amplified and identified as Aspergillus versicolor (GenBank Accession No. OK357263).The voucher sample was maintained in the School of Pharmaceutical Sciences, South-Central Minzu University at À80 C. The Army Military Medical University gave us drug-resistant bacteria as gifts.

Fermentation, extraction, and isolation
The fungus HYQZ-215 was incubated on PDA medium at 28 C for 5 days, then cut into small pieces and inoculated into flasks (rice 80 g, distilled water 80 mL, protein 5&) to culture at 28 C for 40 days.After that, the cultures were extracted with EtOH for seven times and evaporated under reduced pressure, and then extracted four times with EtOAc (1:1, v/v) to get the crude extract (37.4 g).The EtOAc extract was subjected to silica gel column chromatography (CC) eluting with CH 2 Cl 2 -MeOH (100:1-0:100, v/v) to afford three fractions (Fr.A-Fr.C).

Computational methods
A preliminary conformational search was performed in Conflex 6.7 using the MMFF94s method.The frequency calculations and energies of the low-energy conformers were calculated by time-dependent density functional theory (TD-DFT) at the B3LYP/6-31G (d, p) level in methanol with the IEFPCM model.The ECD spectra were generated by the program SpecDis 1.71 using a Gaussian band shape with 0.3 eV exponential half-width from dipole-length dipolar and rotational strengths.

Antibacterial assay
Eight important drug-resistant bacterial strains in clinic were used to evaluate the antibacterial activities of compounds 1-9 by microbroth dilution method.DMSO and ciprofloxacin were used as the negative and positive controls, respectively.Detailed experimental operations can be found in previous study [24].

Antifungal assay
Compounds 1-9 were also evaluated for antifungal activities against seven agricultural pathogenic fungi.Ketoconazole and DMSO were used as the positive and negative controls, respectively.The detailed experimental operations were described previously [24].

Disclosure statement
No potential conflict of interest was reported by the author(s).

Compound 1
was isolated as yellow oil.The molecular formula of C 19 H 25 N 3 O 4 was established by positive HRESIMS data at m/z 360.1915 [M þ H] þ , implying the presence of 9 degrees of unsaturation.The conjugated imine group absorption (1686 cm À1 ) was revealed by IR spectrum.The 1 H-NMR data (
quaternary carbons (d C 135.3 and 121.2), while one oxidized quaternary carbon (d C 84.4) and one sp 3 methine (d C 41.1) were absent in 2. Further analysis of the 2D NMR suggested the hydroxylated Ile unit was replaced by transformed Ile moiety in

Figure 3 .
Figure 3.Comparison between calculated and experimental ECD spectra of 1 in MeOH.

Figure 4 .
Figure 4. Comparison between calculated and experimental ECD spectra of 2 in MeOH.
b Measured in CDCl 3 .