Nitrogenous Compounds from the Antarctic Fungus Pseudogymnoascus sp. HSX2#-11

The species Pseudogymnoascus is known as a psychrophilic pathogenic fungus which is ubiquitously distributed in Antarctica. While the studies of its secondary metabolites are infrequent. Systematic research of the metabolites of the Antarctic fungus Pseudogymnoascus sp. HSX2#-11 led to the isolation of one new pyridine derivative, 4-(2-methoxycarbonyl-ethyl)-pyridine-2-carboxylic acid methyl ester (1), together with one pyrimidine, thymine (2), and eight diketopiperazines, cyclo-(dehydroAla-l-Val) (3), cyclo-(dehydroAla-l-Ile) (4), cyclo-(dehydroAla-l-Leu) (5), cyclo-(dehydroAla-l-Phe) (6), cyclo-(l-Val-l-Phe) (7), cyclo-(l-Leu-l-Phe) (8), cyclo-(l-Trp-l-Ile) (9) and cyclo-(l-Trp-l-Phe) (10). The structures of these compounds were established by extensive spectroscopic investigation, as well as by detailed comparison with literature data. This is the first report to discover pyridine, pyrimidine and diketopiperazines from the genus of Pseudogymnoascus.


Introduction
Nitrogenous compounds represent one of the most momentous family of secondary metabolites which are widely distributed in different biological sources [1]. They have been proved to exhibit various biological activities including cytotoxic, anti-inflammatory, antimicrobial activities and so on [1][2][3][4]. For example, pegaharine D, a β-carboline alkaloid isolated from the seeds of Peganum harmala exhibited strong antiviral activity against herpes simplex virus-2 [5]. For another example, asperversiamides A-C, a kind of cycloheptapeptide, showed potent inhibitory activity against Mycobacterium marinum [6]. Antarctica as the southernmost point of the earth, has the most hostile environment including cold, dry climate and low level of nutrition [7]. Microbes, especially fungi, have been proved to have the potential capacity to produce abundant novel compounds to adapt the extreme habitat. There were more and more bioactive natural products with novel structures have been discovered from Antarctic fungi [8][9][10][11]. The species Pseudogymnoascus is known as a psychrophilic pathogenic fungus with a ubiquitous distribution in Antarctica. While the rare research about its secondary metabolites suggested the potentials to discover interesting compounds [12]. Pseudogymnoascus sp. HSX2#-11, an Antarctic fungus derived from a soil sample of the Fields Peninsula, which can produce various compounds according to our previous study [13], was further investigated to search for new secondary metabolites. As a result, one new pyridine derivative, 4-(2-methoxycarbonyl-ethyl)-pyridine-2-carboxylic Fields Peninsula, which can produce various compounds according to our previous study [13], was further investigated to search for new secondary metabolites. As a result, one new pyridine derivative, 4-(2-methoxycarbonyl-ethyl)-pyridine-2-carboxylic acid methyl ester (1), together with one known pyrimidine, thymine (2) and eight known diketopiperazines (3-10) (Figure 1), were isolated and identified from the potato dextrose agar (PDA) culture broth fermentative extracts of this strain. This paper addresses the isolation, structure elucidation, and bioactivity evaluation of the isolated compounds.

Fungal Materials
The fungus Pseudogymnoascus sp. HSX2#-11 was isolated from a soil sample of the Fields Peninsula at Chinese 35th Antarctic expedition in 2019. The strain was deposited in the State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, China, with the GenBank (NCBI) accession number MT367223.

Antibacterial and Cytotoxic Activity Assays
The antibacterial activities were evaluated by the conventional broth dilution assay [23]. Four pathogenic bacteria, Escherichia coli, Staphylococcus aureus, P. aeruginosa and Bacillus subtilis, and nine marine fouling bacteria, P. fulva, Aeromonas hydrophila, A. salmonicida, Vibrio anguillarum, V. harveyi, Photobacterium halotolerans, P. angustum, Enterobacter cloacae and E. hormaechei, were used, and cipofloxacin was used as a positive control.
The cytotoxicities against human breast cancer (MDA-MB-231, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, L-15), colorectal cancer (HCT116, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, McCOY's 5A), lung carcinoma (A549, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, F-12), pancreatic carcinoma (PANC-1, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, DMEM) and hepatoma (HepG2, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, DMEM) cell lines were evaluated using the sulphorodamine B (SRB) method [24]. Adriamycin was used as a positive control.

Conclusions
In summary, one new pyridine derivative, 4-(2-methoxycarbonyl-ethyl)-pyridine-2carboxylic acid methyl ester (1), together with one pyrimidine, thymine (2), and eight diketopiperazines 3-10, were isolated from the Antarctic fungus Pseudogymnoascus sp. HSX2#-11. All the isolated compounds showed no antibacterial or cytotoxic activities. More bioactivity evaluating models should be needed to find the effects of these secondary metabolites. This is the first time to find pyridine, pyrimidine and diketopiperazines from the genus of Pseudogymnoascus. Our chemical investigation of the Antarctic fungus Pseudogymnoascus sp. HSX2#-11 enriches the chemical diversity of this fungal species.
Supplementary Materials: The following are available online, NMR and HRESIMS spectra of the isolated compounds 1-10: Figure S1