Benzoquinone and furopyridinone derivatives from the marine-derived fungus Talaromyces sp. MCCC 3A01752

Abstract Two new compounds, compounds 1 and 2, were obtained from the culture of a marine-derived fungus Talaromyces sp. MCCC 3A01752, together with 13 known compounds (3–15). Their structures were elucidated based on detailed analysis of NMR, HRESIMS, ECD spectra and OR value. Compound 1 exhibited antibacterial potential against Staphylococcus aureus with a MIC value of 100 μM and cytotoxic activity against gastric cancer cell line MKN1 with a IC50 value of 78.0 μM. Graphical Abstract


Introduction
Marine fungi have been proved to be an important source of natural products with diverse structures and a broad spectrum of bioactivities.Among them, the ascomycetous fungus Talaromyces has been known to produce secondary metabolites with interesting molecular structures, for example, oligophenalenone dimers (Zang et al. 2016) and hybrid phenalenone dimers from Talaromyces stipitatus (Chaudhary et al. 2020), nonadrides from Talaromyces sp.HDN1820200 (Zhao et al. 2021), polycyclic meroterpenoids from Talaromyces sp.CX11 (Cao et al. 2019), and phenyl-substituted phthalides from Talaromyces pinophilus AF-02 (Zhai et al. 2015).
Density-functional-theory (DFT) is an important theory to calculate NMR and ECD data for natural products.Comparison of the calculated NMR data with the experimental can establish the relative configuration of natural products.DFT calculations of ECD spectra can further assign the absolute configuration.Recent years have witnessed an increase in solving structural validation of natural products via DFT approaches (Pescitelli and Bruhn 2016).
During the process for novel biologically active compounds from fungal source, we obtained a fungal strain, Talaromyces chloroloma MCCC 3A01752, which demonstrated antibacterial potential in preliminary screening.Detailed investigation for bioactive constituents from the extract resulted in the isolation of two new compounds (1 and 2), together with 13 known compounds (3-15) (Figure 1).Structural elucidation of new compounds was approached by extensive spectroscopic methods coupled with DFT calculation of ECD spectra.Known compounds were assigned by comparison of spectroscopic data with those reported in literature.The antimicrobial and cytotoxic activities of all the compounds were also evaluated in current study.
Compound 1 was isolated in the form of a yellow amorphous solid.The HRESIMS data revealed the molecular formula was C 11 H 14 O 4 with five degrees of unsaturation, which was deduced from the HR-ESI-MS data (m/z 209.0804 [M -H] -; calcd.for 209.0808,C 11 H 13 O 4 , D À 1.9 ppm) (Figure S1).The carbon types were determined by interpretation of 13 C NMR, HSQC and DEPT spectra as three methyl units (Figures S3-S5), one methylene unit, one oxygenated methine unit, and six sp 2 nonprotonated carbons including two ketone carbonyls and one oxygenated olefinic carbons.The NMR data suggested a tetrasubstituted benzoquinone moiety in 1, accounted for all the unsaturation.HMBC correlations of OH!C-4/C-5/C-6 (Figure S6) together with the chemical shifts of C-5 (d C 150.7) and C-6 (d C 117.2) placed the OH group at C-5, and the remaining nonprotonated carbons should be at d C 117.2, d C 136.9 and d C 145.3.Interpretation of HMBC correlations of H 3 -7!C-5/C-6/C-7 and of H 3 -8!C-1/C-2/C-3 (Figure S6) demonstrated that C-2 and C-6 were methyl-bearing.Furthermore, COSY correlations of H 2 -9!H-10!H 3 -11 (Figure S7), doublet at d H 2.69, sextet at d H 3.99 and doublet at d H 1.27 (Figure S2), allowed the assignment of 2-hydroxypropanyl group, which attached to C-3 based on HMBC correlations of H 2 -9!C-2/C-3/C-4 (Figure S6).The opposite sign of optical rotation between compound 1 and the reported anserinone B (Wang H et al. 1997a(Wang H et al. , 2004b) ) suggested the S stereochemistry at the separated stereocenter in compound 1, and we finally establish the absolute configuration to be 10S and named anserinone C.
The antimicrobial activities of compounds 1-15 was evaluated using strains of Staphylococcus aureus CICC 10384, Vibrio parahaemolyticus VP-HL, Vibrio parahaemolyticus ATCC 17802, and Escherichia coli CICC 10302.Compound 1 showed inhibitory activities against Staphylococcus aureus CICC 10384 with a MIC value of 100 lM (positive control: chloramphenicol, MIC: 15.5 lM), while other compounds displayed no antibacterial activities.We then evaluated the cytotoxicity of 1-15 against the gastric cancer cell line MKN1.Compound 1 exhibited growth inhibition effects with IC 50 values of 78.0 lM, respectively.Other compounds displayed weak cytotoxic activities with IC 50 values over 100 lM (positive control: cisplatin, IC 50 : 8.8 lM).

General experimental procedures
The spectroscopic experiments of NMR, UV, MS and OR were identical to those reported previously (Hong et al. 2022).

Fungal material
Strain HM4-3-T3 of Talaromyces sp. was identified by ITS sequence and deposited at the Marine Culture Collection of China (MCCC) with preservation number MCCC 3A01752.The strain HM-4-3-T3, collected from the sea water from Dongshan Island in Fujian Province of China, shared the highest ITS similarity (94.31%) with two type strains Talaromyces coalescens CBS:103.83 and Talaromyces pittii CBS:139.84.The ITS sequence of the fungus has been submitted to GenBank with the Accession No. ON926872.

ECD calculation
The conformational search was carried out using the procedure similar to those previously reported.The ECD spectrum was calculated at Cam-B3LYP/Def2SVP level following the procedure reported previously (Wang W et al. 2018a(Wang W et al. , 2020b(Wang W et al. , 2020c;;Hong et al. 2022).

Antimicrobial and cytotoxic assay
Strains of Staphylococcus aureus CICC 10384, Vibrio parahaemolyticus VP-HL, Vibrio parahaemolyticus ATCC 17802, and Escherichia coli CICC 10302 were used for in vitro antimicrobial assay, following the protocol which was reported previously (Hong et al. 2022).The human gastric cancer cell line MKN1 was purchased from American Type Culture Collection (ATCC, LGC Standards SLU, Barcelona, Spain).The cell viability (CCK8 assays) was evaluated and the detailed were described previously (Hong et al. 2022).

Conclusion
In summary, we have isolated two new compounds, anserinone C (1) and (R)-3hydroxy-2,7-dimethylfuro[3,4-b]pyridin-5(7H)-one (2), together with 13 known compounds (3-15).The absolute configurations of new compounds were approached by analysis of OR value and ECD calculation, respectively.All the compounds were subjected to antibacterial and cytotoxic assay, and anserinone C (1) exhibited antibacterial potential against Staphylococcus aureus and cytotoxic activities against gastric cancer cell line MKN1.Anserinone C (1) differed from the previously reported anserinone A and anserinone B (Wang H et al. 1997a(Wang H et al. , 2004b) ) in the attachment of a methyl to C-6, which enriched the chemical diversity of benzoquinone derivatives from filamentous fungi.