Polyketides with potential bioactivities from the mangrove-derived fungus Talaromyces sp. WHUF0362

Metabolites of microorganisms have long been considered as potential sources for drug discovery. In this study, five new depsidone derivatives, talaronins A-E (1–5) and three new xanthone derivatives, talaronins F–H (6–8), together with 16 known compounds (9–24), were isolated from the ethyl acetate extract of the mangrove-derived fungus Talaromyces species WHUF0362. The structures were elucidated by analysis of spectroscopic data and chemical methods including alkaline hydrolysis and Mosher’s method. Compounds 1 and 2 each attached a dimethyl acetal group at the aromatic ring. A putative biogenetic relationship of the isolated metabolites was presented and suggested that the depsidones and the xanthones probably had the same biosynthetic precursors such as chrysophanol or rheochrysidin. The antimicrobial activity assay indicated that compounds 5, 9, 10, and 14 showed potent activity against Helicobacter pylori with minimum inhibitory concentration (MIC) values in the range of 2.42–36.04 μmol/L. While secalonic acid D (19) demonstrated significant antimicrobial activity against four strains of H. pylori with MIC values in the range of 0.20 to 1.57 μmol/L. Furthermore, secalonic acid D (19) exhibited cytotoxicity against cancer cell lines Bel-7402 and HCT-116 with IC50 values of 0.15 and 0.19 μmol/L, respectively. The structure–activity relationship of depsidone derivatives revealed that the presence of the lactone ring and the hydroxyl at C-10 was crucial to the antimicrobial activity against H. pylori. The depsidone derivatives are promising leads to inhibit H. pylori and provide an avenue for further development of novel antibiotics. Supplementary Information The online version contains supplementary material available at 10.1007/s42995-023-00170-5.


Introduction
Microbial secondary metabolites have received great attention as a potential resource of lead drugs owing to their productive biological activities and massive chemical diversity (Hai et al. 2021;Xu et al. 2022). Due to the special mangrove Edited by Chengchao Chen. environment, including high salinity, low oxygen, nutrient limitation, and drought, mangrove-derived fungi have the biosynthetic potential to produce a variety of unique secondary metabolites (Liang et al. 2019;Nathan et al. 2020;Xu et al. 2014;Zhang et al. 2021). The genus Talaromyces (Trichocomaceae) is a sexual state of Penicillium, and has the potential to produce depsidones (Zhao et al. 2015;Wu et al. 2015). In the viewpoint of ecologies, the occurrence of Talaromyces makes these fungi increasingly regarded as a source of interesting bioactive compounds, leading to the discovery of drugs, such as penicillin, compactin, antimycotoxins, and miscellaneous antitumor products (Nicoletti and Trincone 2016;Nicoletti et al. 2018).
The depsidones were a series of compounds derived from depsides by a loss of hydrone in an oxidative cyclization and aroused great pharmacological interest as antimicrobial and cytotoxic agents (Hong et al. 2018;Ureña-Vacas et al. 2022;Yilmaz et al. 2004). Some depsidones act as RecA protein inhibitors by increasing bactericidal activity and reducing antibiotic resistance. Furthermore, depsidones have also targeted the protein FabZ of the bacterial system for fatty acid biosynthesis (FAS) (Alam et al. 2016;McGillick et al. 2016). Depsidones can attenuate cell tumor growth by acting as selective inhibitors of Plk1 activity or directly target antiapoptotic Bcl-2 family proteins (Hong et al. 2018;Wil-liaNms et al. 2011).
In the current study, the fungus Talaromyces sp. WHUF0362, isolated from the mangrove soil sample collected from Yalog Bay, at Sanya, Haian, China, showed potent antimicrobial activities against Escherichia coli and H. pylori G27. During our search for active secondary metabolites from the marine-derived fungi, the chemical investigation of secondary metabolites of Talaromyces sp. WHUF0362 was performed. This work resulted in the purification and identification of five new depsidones, talaronins A-E (1-5), and three new xanthone derivatives, talaronins F-H (6-8), together with 16 known compounds (9-24) (Fig. 1). In addition, the isolated compounds were evaluated for antimicrobial activity (ten Gram-negative bacteria, seven Gram-positive bacteria, a Mycobacterium, and two fungi) and cytotoxic activity (Bel-7402, HCT-116, and A549).

Results and discussion
The strain Talaromyces sp. WHUF0362 was isolated from the mangrove soil sample collected from Yalog Bay, at Sanya, Haian, China. A crude extract of Talaromyces sp. WHUF0362 cultivated in the PDA medium exhibited antimicrobial activities against Escherichia coli and H. pylori G27. A further chemical investigation of the rice fermentation 1 3 products of Talaromyces sp. WHUF0362 was carried out and led to isolation and identification of 24 polyketide derivatives, eight (1-8) of which were determined as new by comprehensive analysis of spectroscopic data (1D and 2D NMR, HRESIMS, IR, and UV) and chemical methods including alkaline hydrolysis and Mosher's method.
Talaronin C (3) was obtained as a colorless oil with the molecular formula C 25 H 28 O 8 inferred from its HRESIMS (m/z 474.2139 [M + NH 4 ] + , calcd. for C 25 H 32 NO 8 : m/z 474.2122), accounting for twelve degrees of unsaturation. The general features of the 1 H and 13 C NMR spectra resembled those of talaromyone B (12) (Cai et al. 2017) except for the presence of an additional acetyl moiety. The significant downfield shift of H-10 and the key correlations from H-10 (δ H 5.35, 5.32) to a carbonyl group at δ C 172.1 suggested the acetyl group should be located at C-10. Thus, the structure of 3 was determined as shown and given the name talaronin C.
Talaronin D (4) was isolated as a yellow oil with the molecular formula of C 21 H 22 O 6 based on its HRESIMS data (m/z: 369.1337 [M-H] -, calcd. for C 21 H 22 O 6 : m/z 369.1344), accounting for eleven degrees of unsaturation. Analyses of the 1 H and 13 C NMR (Table 1) signals of 4 indicated the presence of an isoamyl (δ H 5.07, 1.63, 1.43, 1.78, 0.97, 0.94; δ C 66.9, 47.6, 25.1, 21.9, 23.5), an aldehyde group (δ H 10.61, δ C 187.8), and a methoxyl (δ H 3.91, δ C 63.2). The 1D NMR spectroscopic data of 4 were similar to those obtained from 1 beyond the absence of a dimethyl acetal unit and the presence of an aldehyde group in 4. The aldehyde group was determined to be at C-10 as evident by HMBC correlations from H-10 (δ H 10.61) to C-5 (δ C 125.4) and C-10 (δ C 128.6). Unambiguously, the structure of 4 was assigned as shown and named to be talaronin D.
Talaronin E (5)  , an ester group (δ C 167.5) and a acetyl (δ H 2.03, δ C 21.3, δ C 170.4). These groups were attributed to the eleven degrees of unsaturation given by HRESIMS, which indicated that there was no lactonic ring in 5 compared to the depsidone. 1D NMR spectra of 5 showed a close similarity with those of secopenicillide B (14) (Komai et al. 2006) with exceptions of the absence of the methoxyl group and the presence of an ethoxyl [δ H 4.53 (q, J = 7.1 Hz) 1.47 (t, J = 7.2 Hz)]. The HMBC correlation from δ H 4.53 to C-9 (δ C 167.5) suggested the ethoxyl moiety was bonded to the C-9 carbonyl. Therefore, compound 5 was named as talaronin E.
Talaronin F (6) was isolated as a brown oil with a molecular formula of C 16 H 14 O 8 revealed by analysis of its HRESIMS (m/z 333.0635 [M-H] -, calcd. for C 16 H 13 O 8 : m/z 333.0616), implying ten degrees of unsaturation in 6. Comparison of the 1 H NMR and 13 C NMR data of 6 with those of ergochrome E (Yong et al. 2021) hinted 6 should be an xanthone analogue of ergochrome E. The 1D NMR data (Table 2) were fully assigned by detailed analysis of the HSQC and HMBC spectra. The presence of three aromatic protons [δ H 7.50 (t, J = 8.3 Hz), 6.63 (d, J = 8.2 Hz), 6.61 (d, J = 8.3 Hz)] indicated an AMX-spin system in 6. The HMBC correlations from H 3 -11 (δ H 2.17) and H-5 (δ H 4.65) to C-6, 7 (δ C 156.5, 128.4) denoted the aliphatic carbons of C-5 and C-6 in ergochrome E were replaced by two olefinic carbons in 6. The chemical shift of δ C 191.2 in 6 implies an α, β-unsaturated ketone by analysis of the chemical shift of C-6 and C-7 combined with the weak  HMBC correlation of H 3 -11/C-8. The HMBC correlation from H-7 to C-8a signified a hydroxyl at C-8a. Thus, compound 6 was named talaronin F. Talaronin G (7) was obtained as a yellow solid. The molecular formula was determined to be C 16 H 12 O 8 based on an ion at m/z 331.0458 [M-H]by the HRESIMS, indicating eleven degrees of unsaturation. The 1 H NMR (Table 2) spectrum exhibited an AMX-spin system [δ H 7.50 (dd, J = 7.8, 0.9 Hz), 7.30 (t, J = 8.0 Hz), 7.06 (dd, J = 7.8, 0.9 Hz)], two overlapping aromatic proton signals (δ H 6.90), and a methoxyl (δ H 3.72). The 13 C NMR data revealed the presence of three carboxyl groups, two aromatic rings, and a methoxyl by taking the 1 H NMR and HSQC spectroscopic data into account. The general features of the 1 H and 13 C NMR spectra of 7 were similar to those of the known compound methyl peniphenone ). The main difference was that there were two overlapping aromatic proton signals in 7 instead of the AMX-spin system, which was in accordance with a symmetrically substituted aromatic ring. The HMBC correlation from H-5, 7 (δ H 6.90) to a carbonyl resonance (δ C 175.5) suggested the carboxyl was located at C-6 combined with the analysis of HRESIMS data. Hence, the structure of 7 was established as shown and assigned the name talaronin G.
Talaronin H (8), a yellow solid, was found to possess a molecular formula of C 17 H 14 O 9 from HRESIMS ion at m/z 361.0547 [M-H] -(Calcd. for C 17 H 13 O 9 , 361.0565). The 1 H and 13 C NMR data (Table 2) suggested the presence of a characteristic ketone carboxyl, two aromatic rings, and two methoxy groups. 1D NMR spectrum of 8 was also in accordance with 7 except for an additional methoxyl and the absence of the AMX-spin system. The HMBC correlations between H-3-Me (δ H 3.85) and C-3 (δ C 162.2), H-4 (δ H 6.98)/H-2 (δ H 6.63) and C-3 (δ C 162.2) indicated the methoxy group was placed at C-3. The structure of 8 was identified as shown and named talaronin H.
The known compounds were identified as purpactin A (9)  Compared with the reported depsidone derivatives, the main differences of the five new depsidone derivatives are in the different substituents of C-10, C-9, and C-1′. The known depsidones usually have an aldehyde group at C-10. However, compounds 1 and 2 each contain a dimethyl acetal group at C-10 and the aldehyde group in compound 3 was reduced to a hydroxymethyl group. This provided clues for us to speculate on the biosynthetic pathway of the depsidone. The skeleton of depsidones was produced from acetyl-and malonyl-coenzyme A by nonreducing polyketide synthase (PKS) (Cox 2007;Xu et al. 2014). It was accepted that the depsidones involved the oxidative coupling of benzophenone to give spirobenzofuran-1,2′-cyclohexa-3′,5′-diene-2′,3-dione as an intermediate, which in turn rearranged to the depsidone (Nishida et al. 1992;Xu et al. 2014). Most of the isolates herein we obtained could be divided into two series, the depsidone derivatives (1-5 and 9-18) and the xanthone derivatives (6-8 and 19). Both groups probably had the same biosynthetic precursors such as chrysophanol or rheochrysidin. The oxidation product of chrysophanol/ rheochrysidin was methylated to offer compounds 6 and 7 (Wei and Matsuda 2020). Cyclization of the oxidation product gave compounds 6 and 18 (Frisvad et al. 2020) or under the action of dimerase to obtain compound 19 ). In addition, the intermediate of spirane was obtained by PKS (Xu et al. 2014), and the rearrangement occurred due to the instability of the spiroane structure (Nishida et al. 1991). The isoprenylation of the rearrangement product was then methylated and/or acetylated to give compounds 1-3 and 10-12 or directly methylated and/or acetylated to yield compounds 5 and 13-17 (Kikuchi et al. 2012;Masters and Bräse 2012). The proposed biogenetic relationship of the isolated metabolites was shown in Supplementary Fig. S68.
The biosynthetic pathways of 3-5 were expected to be the same as that of 1. Thus, the absolute configuration of C-1′ in 3-5 was proposed to be S.
All of the isolates with adequate amount were evaluated for their antimicrobial activities and cytotoxicity activities (Supplementary Table S1, Table S2, and Table S3). Compounds 5, 9, 10, and 14 showed antibacterial activity against H. pylori with MIC values in the range from 2.42 to 36.04 μmol/L, with amoxicillin as positive control with MIC values of 0.14 to 38.14 μmol/L. Compound 11 showed antibacterial activity against Staphylococcus aureus NEW-MAN with an MIC value of 38.83 μmol/L (Table 3). Particularly, compound 19 showed significant antimicrobial activity against H. pylori with MIC values of 0.20 to 1.57 μmol/L. In addition, compound 19 preeminently inhibited cancer cell lines Bel-7402 and HCT-116 with IC 50 values of 0.15 and 0.19 μmol/L compared with 5-fluorouracil as a positive control with IC 50 values of 13.69 and 12.23 μmol/L, respectively. According to their structural characteristics, the isolated depsidone derivatives could be divided into two categories: with lactone ring and without lactone ring. Interestingly, 5 and 13-17 without lactone ring exhibited the weak anti-H. pylori activity with MIC values higher than 32.65 μmol/L. While 9 and 10 possessed the lactone ring showed anti-H. pylori activity with MIC values of 2.41 to 10.75 μmol/L. It was suggested that the presence of the lactone ring in depsidone derivatives was related to the anti-H. pylori activity. Furthermore, 4 displayed no activity against H. pylori; whereas 10, reduzate of 4, had better anti-H. pylori activity with MIC values of 5.38 to 10.75 μmol/L. Additionally, the substituted group at C-10 in 9 was a hydroxyl which made it exhibit better anti-H. pylori activity with MIC values of 2.41 to 4.83 μmol/L. The above evidence indicated that the presence of the lactone ring and the hydroxyl at C-10 played an important role for antimicrobial activity against H. pylori. A previous study (Cai et al. 2017) indicated talaromyone B (12) possessed inhibitory activity against Bacillus subtilis. This work first presented the inhibitory activity against H. pylori of the depsidone analogues and provided an avenue for the further development of novel antibiotics.

Conclusions
In summary, five new depsidones, talaronins A-E (1-5), and three new xanthone derivatives, talaronins F-H (6-8), together with 16 known compounds were isolated from the culture of the mangrove-derived fungus Talaromyces sp. WHUF0362. Most of the isolates, could be divided into two series of compounds, the depsidone derivatives (1-5 and 9-17) and the xanthone derivatives (6-8, 18 and 19), and all of them probably had the same biosynthetic precursors, chrysophanol or rheochrysidin. In the bioactivity assays, secalonic acid D (19) demonstrated promising inhibitory activity against the cancer cell lines Bel-7402 and HCT-116 with IC 50 0.15 and 0.19 μmol/L, respectively. In additional, secalonic acid D (19) showed significant antimicrobial activity against four strains of H. pylori with MIC values of 0.20 to 1.57 μmol/L. In addition, the investigated isolates 5, 9, 10, and 14 showed potential activity against H. pylori with MIC values of 2.42 to 36.04 μmol/L. The structure-activity relationship of depsidones revealed that the presence of the lactone ring and the hydroxyl at C-10 was crucial to the antimicrobial activity against H. pylori. These promising biological findings could provide an optimistic direction for finding new drugs against H. pylori.

General Experimental Procedure
All the 1D and 2D NMR spectra were obtained by a Bruker AVANCE III 600 MHZ spectrometer with TMS as an internal standard (Bruker company, Switzerland). The HRESIMS data were obtained on an Agilent 6210 TOF MS system (Agilent Technologies, Santa Clara, CA, USA) or AB SCIEX Triple TOF 5600 + (AB SCIEX, USA). Optical rotations were measured by a JASCO P-1020 polarimeter (Jasco Tokyo Japan). UV spectra were performed in MeOH by using a Shimadzu UV spectrometer-1800 (Shimadzu Corp., Kyoto, Japan). IR spectra (KBr) were obtained on a Nicolet 6700 FT-IR spectrometer (Thermo Electric Nicoli, United States). Semipreparative high performance liquid chromatography (HPLC) was performed by an Agilent 1260 separation system with an Aglient ZORBAC SB-C 18 column (5 μm, 250 mm × 9.4 mm, 3 mL/min). Sephadex LH-20 gel (GE Healthcare, Uppsala, Sweden) and MCI gel (Mitsubishi Chemical Corp., Japan) were used in column chromatography. And silica gel (200-300 mesh for column chromatography, GF254 for TLC) was supplied by the Yantai Zhifu Huanwu Silicone Factory, Yantai, China.

Fungal material
The fungal strain Talaromyces sp. WHUF0362 was isolated from a mangrove soil sample collected from Yalog Bay, at Sanya, Haian, China, in Dec, 2018. The strain was selected by strong and selective activity against microbial pathogens during assays against E. coli CCTCC AB 93,154, S. aureus CCTCC AB 91,093 and Candida albus CCTCC AY 206,001, and presented serious peaks at UV

Extraction and Isolation
Fermentation products were extracted three times with EtOAc by soaking overnight. The crude extraction (83.77 g) was obtained by vacuum distillation. This extract was fractionated by silica gel column chromatography using the PE (petroleum ether, 60-90 °C) and the EtOAc gradient system (1:0 to 0:1, v/v) to give 7 fractions (A-G). Fraction D was applied to the silica gel column chromatography eluting with a step gradient of petroleum ether (PE):EtOAc (10:1 to 0:1, v/v) to obtain 9 fractions (D1-D9