A new quinolone and acetylcholinesterase inhibitors from a sponge-associated fungus Penicillium sp. SCSIO41033

Abstract The chemical investigation of the EtOAc extract from the solid rice medium cultured with a sponge-associated fungus Penicillium sp. SCSIO41033 led to the isolation of two quinolones including a new one, penicinolone (1), three xanthone derivatives (3–5), and four anthraquinones (6–9). Their structures were determined by comprehensive analysis of 1H and 13C NMR, COSY, HSQC, and HMBC spectroscopic, and HRESIMS mass spectrometric data. The bioactive assays revealed that compounds 1 and 2 showed no antimicrobial activities against five bacteria and eight fungi, and compounds 5, 8 and 9 exhibited inhibition against AChE with IC50 values of 45.9, 42.5 and 40.5 μg/mL. Molecular docking analysis was performed to explore the interactions between active molecules and AChE protein, which indicated that xanthone and anthraquinone derivatives had the potential for developing AChE inhibitors. Graphical Abstract


Introduction
Acetylcholine is an important neurotransmitter related to learning and memory in the nervous system (Nordberg 2006). In the pathogenesis of Alzheimer's disease, a neurodegenerative disorder among the elderly, the neuropathological evidence have demonstrated that cholinergic functions declined in the basal forebrain and cortex in senile dementia (Whitehouse et al. 1982). So far, there are only several clinical drugs to treat Alzheimer's disease, most of which are acetylcholinesterase (AChE) inhibitors, like donepezil, rivastigmine, rivastigmine, and huperzine A (Winblad et al. 2006;Wen et al. 2009;Mangialasche et al. 2010). In order to discover more effective AChE inhibitors, a series of fungal metabolites with significant 50% inhibiting concentration (IC 50 ) values against AChE were obtained from marine fungi, such as sporothrin A (IC 50 1.05 mM) from Sporothrix sp. #4335 (Wen et al. 2009), 13-hydroxypalitantin (IC 50 12.3 nM) from Penicillium sp. (Huang et al. 2014), infractopicrin and 10-hydroxy-infractopicrin (IC 50 9.7 and 12.7 mM) from Cortinarius infractus Berk (Geissler et al. 2010). As part of our ongoing research for bioactive natural products as AChE inhibitors from marine fungi (Han et al. 2020;Long et al. 2021;Pang et al. 2021), the sponge-associated fungus Penicillium sp. SCSIO41033 was studied. Two quinolones including a new one, penicinolone (1), three xanthone derivatives (3-5), and four anthraquinones (6-9) (Figure 1) were obtained from its EtOAc extract of the solid rice medium.
Quinolone alkaloids, known as antibiotics, play a key role in the pharmaceutical and agrochemical fields. The two quinolone alkaloids (1 and 2) were screened for antibiotic activities against five bacteria, Staphylococcus aureus ATCC 29213, Acinetobacter baumannii ATCC 19606, Klebsiella pneumonia ATCC 13883, Enterococcus faecalis ATCC29212 and Escherichia coli ATCC 25922, and eight fungi, Acternaria alternate, Colletotrichum acutatum HNM RC178, Colletotrichum asianum HNM 408, Colletotrichum gloeosporioides HNM 1003, Curvularia australiensis, Fusarium oxysporum HNM1003, Pyricularia oryaza HNM1003 and Rhizoctonia solani. However, both of them displayed no inhibitions against all microbes. Furthermore, compounds 1-9 were evaluated for the inhibitory activities against AChE. Compounds 1-5, 8 and 9 exhibited inhibitory rates between 42.5% and 65.8% under the concentration of 50.0 lg/mL, while compounds 6 and 7 showed inhibitory rates below 40%. The IC 50 values of the compounds with inhibitory rate over 50% were measured as shown in Table S1. The bioactive assay and enzyme inhibitory assay were conducted as described in the literature ).
Subsequently, compounds 4, 5, 8 and 9 were selected for molecular docking analysis to explore the interactions between active compounds and AChE. The crystal structure of AChE (PDB ID: 4EY7) was retrieved from the Protein Data Bank (http://www.rcsb.org/ pdb) for the molecular docking study, which was conducted by the AutoDock vina program (Trott and Olson 2010;Vald es-Tresanco et al. 2020). As shown in Figure S9, all four compounds could tightly bind into the active pocket of AChE with the calculated binding affinity of À9.7, À10.1, À9.7 and À10.3 kcal/mol, respectively. For xanthone derivatives (4 and 5), one additional hydroxyl group could increase the anti-AChE activities based on the enzyme inhibitory experiment and molecular docking results. Two hydrogen bonds were formed between 8 and residues TYR-337 and HIS-447, while four hydrogen bonds were formed between 9 and residues TYR-124, GLY-120 and SER-125, which suggested that one more hydroxyl group could help active molecules to form more strong interactions with AChE. The above results told us that xanthone and anthraquinone derivatives possessed the potential to develop AChE inhibitors.

General experimental procedures
1D and 2D NMR spectra were measured on a Bruker AVANCE HD 700 MHz NMR spectrometer with tetramethylsilane as an internal standard. HRESIMS spectra were recorded on a Bruker maXis Q-TOF mass spectrometer in positive ion mode. UV spectra were recorded on a UV-2600 UV-vis spectrophotometer (Shimadzu). HPLC was carried out on a Hitachi Primaide with YMC ODS Series column (YMC-Pack ODS-A, YMC Co. Ltd., 250 Â 10 mm i.d., S-5 lm, 12 nm). The TLC plates with silica gel GF254 (0.4-0.5 mm, Qingdao Marine Chemical Factory) were used for analysis and preparative TLC. Column chromatography was carried out on silica gel (200-300 mesh, Jiangyou Silica Gel Development Co.), Sephadex LH-20 (40-70 lm, Amersham Pharmacia Biotech AB), and YMC Gel ODS-A (12 nm, S-50 lm YMC). Spots on TLC were detected under UV light or by heating after spraying with the mixed solvent of saturated vanillin and 5% H 2 SO 4 in H 2 O. Sea salt (Guangzhou Haili Aquarium Technology Company) was a commercial product.

Fungal material
The fungal strain SCSIO41033 was isolated from a marine sponge sample that was collected from zhongsha islands. The internal transcribed spacer (ITS) region of the strain SCSIO41033 was amplified by polymerase chain reaction process and the result sequence has been submitted to the GenBank database (https://www.ncbi.nlm.nih. gov) with accession No. OM84515. The ITS sequence showed that it shared a significant homology (100%) to that of Penicillium citrinum NRRL 1841 by nucleotide basic local alignment search tool (accession No. NR121224.1), so it was designated as Penicillium sp. and was named as Penicillium sp. SCSIO41033. The producing strain was deposited at the Key Laboratory of Tropical Marine Bioresources and Ecology, Chinese Academy of Sciences.

Molecular docking simulation
The

Conclusion
In conclusion, one new quinolone derivative penicinolone (1) along with eight known compounds (2-9) were obtained from the solid rice medium of a sponge-associated fungus Penicillium sp. SCSIO41033. Quinolones were known as antibiotics, however, both compounds 1 and 2 showed no antimicrobial activities against five bacteria and eight fungi. In addition, compounds 5, 8 and 9 exhibited inhibition against AChE with IC 50 values of 45.9, 42.5, and 40.5 lg/mL. Furthermore, molecular docking simulation suggested that one more hydroxyl substituent of anthone or anthraquinone derivatives could help form more strong interactions with AChE protein. Thus, the comparison between 4 and 5, 8 and 9 indicated that the hydroxyl group could be the key element for the structure-activity relationship of the xanthone or anthraquinone derivatives based on the above result. Our finding not only enriched the diversity of quinolones from fungal sources but also provided some potential molecules for the development of AChE inhibitors.