Bioactive Pyridone Alkaloids from a Deep-Sea-Derived Fungus Arthrinium sp. UJNMF0008

Eight new 4-hydroxy-2-pyridone alkaloids arthpyrones D–K (1–8), along with two known analogues apiosporamide (9) and arthpyrone B (10), were isolated from a deep-sea-derived fungus Arthrinium sp. UJNMF0008. The structures of the isolated compounds were elucidated on the basis of spectroscopic methods with that of 1 being established by chemical transformation and X-ray diffraction analysis. Compounds 1 and 2 bore an ester functionality linking the pyridone and decalin moieties first reported in this class of metabolites, while 3 and 4 incorporated a rare natural hexa- or tetrahydrobenzofuro[3,2-c]pyridin-3(2H)-one motif. Compounds 3–6 and 9 exhibited moderate to significant antibacterial activity against Mycobacterium smegmatis and Staphylococcus aureus with IC50 values ranging from 1.66–42.8 μM, while 9 displayed cytotoxicity against two human osteosarcoma cell lines (U2OS and MG63) with IC50 values of 19.3 and 11.7 μM, respectively.


Introduction
The increasing antibiotic resistance and high spreading rate of pathogenic bacteria have become severe public healthcare threats globally, and the efforts towards new antibacterial drugs remain a pressing task [1]. Marine fungi have been considered to be an invaluable resource for bioactive compounds and play an important role in the search for novel antimicrobial compounds [1][2][3]. Fungi of the genus Arthrinium are widely distributed throughout the world and have proven to produce diverse secondary metabolites with a variety of bioactivities, including cytotoxicity [4][5][6][7], antimicrobial [8], anti-HSV [9], AChE inhibitory [4], COX-2 inhibitory [10], syncytium formation inhibitory [11] and Maxi-K channel modulatory [12] activities, as well as lethality against brine shrimp [13].

Structure Elucidation
Compound 1 was obtained as white powder. The HR-ESIMS ion for 1 at m/z 444.2032 ([M − H] − , calcd. 444.2028) suggested a molecular formula of C24H31NO7, which was 16 amu more than that of its co-metabolite arthpyrone B (10) [4], supportive of an oxygenated analogue. Detailed analysis of the 1 H and 13 C NMR data (Tables 1 and 2) indicated that 1 incorporated a skeleton similar to that of arthpyrone B (10) [4], with a likely additional ester group as supported by the NMR differences of the remarkably upfield shifted C-13 resonance (from 208.4 in 10 to 172.9 in 1) and of adjacent C-9, C-14, C-15, C-17 and C-19 signals. This deduction was further confirmed by inspection of 2D NMR data. The COSY correlations ( Figure 2) of 1 revealed two spin coupling systems from H2-1-H-10 (including branch fragments from H-3-H3-11 and H-8-H3-12) of a decalin moiety and from H-21-H2-25. Subsequent examination of HMBC data ( Figure 2) revealed key correlations from H-17 to C-14, C-15, C-18, C-19 and C-20, H-21 to C-18, C-20 and C-25, H-22 to C-19 and H-9 to C-13, leading to the construction of Fragments A and B that were identical to those in 10. The two fragments were linked via an ester bond as demonstrated by the MS difference (amu 16) and 13 C NMR chemical shift variations for C-13 (ΔδC −35.5) and C-14 (ΔδC 10.9) between 1 and 10. The planar structure of 1 was thus established with an ester bridge between the pyridone and decalin moieties.

Structure Elucidation
Compound 1 was obtained as white powder. The HR-ESIMS ion for 1 at m/z 444.2032 ([M − H] − , calcd. 444.2028) suggested a molecular formula of C 24 H 31 NO 7 , which was 16 amu more than that of its co-metabolite arthpyrone B (10) [4], supportive of an oxygenated analogue. Detailed analysis of the 1 H and 13 C NMR data (Tables 1 and 2) indicated that 1 incorporated a skeleton similar to that of arthpyrone B (10) [4], with a likely additional ester group as supported by the NMR differences of the remarkably upfield shifted C-13 resonance (from 208.4 in 10 to 172.9 in 1) and of adjacent C-9, C-14, C-15, C-17 and C-19 signals. This deduction was further confirmed by inspection of 2D NMR data. The COSY correlations ( Figure 2) of 1 revealed two spin coupling systems from H 2 -1-H-10 (including branch fragments from H-3-H 3 -11 and H-8-H 3 -12) of a decalin moiety and from H-21-H 2 -25. Subsequent examination of HMBC data ( Figure 2) revealed key correlations from H-17 to C-14, C-15, C-18, C-19 and C-20, H-21 to C-18, C-20 and C-25, H-22 to C-19 and H-9 to C-13, leading to the construction of Fragments A and B that were identical to those in 10. The two fragments were linked via an ester bond as demonstrated by the MS difference (amu 16) and 13 C NMR chemical shift variations for C-13 (∆δ C −35.5) and C-14 (∆δ C 10.9) between 1 and 10. The planar structure of 1 was thus established with an ester bridge between the pyridone and decalin moieties.
The relative stereochemistry of 1 was assigned to be identical to that of arthpyrone B (10) based on analyses of NOESY data and 1 H-1 H couplings. More specifically, the key NOESY correlations from  (2.4 Hz). Unfortunately, the stereochemical relationship between Fragments A and B could not be determined due to the interruption of the achiral pyridone unit. However, a further transformation of 10 to 1 (see the Experimental Section) via Baeyer-Villiger oxidation connected the chemical relationship between the two co-metabolites and also confirmed the structural assignment. Finally, a suitable single crystal of 1 was obtained from the MeOH-H 2 O binary system and subject to X-ray diffraction analysis, which not only verified the structure of 1, but also established its absolute stereochemistry ( Figure 3) as 3R, 5S, 8R, 9R, 10R, 20R, 21S, 22S, 23S (Flack parameter, 0.0(3)). Compound 1 was thus unambiguously characterized and was named arthpyrone D, following arthpyrones A−C, isolated from another fungus of the same genus [4]. It is interesting to point out that the assigned C-20 configuration for 1 is opposite that described for arthpyrones A and B [4]. By careful examination of the reported ORTEP view of arthpyrone A, it is clear that both arthpyrones A and B also bear a 20R absolute configuration, while the authors inverted the C-20 stereochemistry by mistake when drawing the structures in two dimensions.    (Figure 4) from H-9 (δH 2.88) to C-13, H-17 (δH 7.31) to C-14, C-15, C-18, C-19 and C-20 and H-21 (δH 3.71) to C-18, C-20 and C-25 were also consistent with those observed for 1. Therefore, the aforementioned MS and NMR differences, the absence of HMBC correlation from H-22 to C-19 and the more polar nature of 2 all supported the planar structure of 2 without the epoxy bridge between C-19 and C-22. The relative configuration of 2 was assigned on the basis of NOESY data (Figure 4). The configuration of the decalin moiety was identical to that of 1 as indicated by NOESY correlations and similar NMR data. The NOESY correlations from H-25b (δH 1.67) to both H-21 (δH 3.71) and H-23 (δH 3.69) suggested their axial and co-planar nature, while those from H-22 (δH 3.62) to both H-17 (δH 7.31) and H-24b (δH 1.54) supported that H-22, H-24b and the pyridone unit were axially located on the other side of the hexane ring. The structure of 2 was hence elucidated as shown in Figure 4.    [14,15]. Analysis of the NMR data (Tables 1 and 2) for 3 revealed the presence of a decalin moiety as indicated by a close resemblance between its NMR data and those of 9, and the main NMR differences between the two co-metabolites observed for the left-side Fragment A. Further inspection of COSY data ( Figure 5) confirmed the existence of the decalin motif and also established the same spin coupling system from H-21-H2-25 as that in 9. In addition, the HMBC correlations ( Figure 5) from H-9 (δH 4.44) to C-13 and C-14 verified the connection between Fragments A and B, while those from H-17 (δH 7.69) to C-14, C-15, C-18, C-19 and C-20, along with those from H-25b (δH 1.51) to C-20 and C-21, enabled the assembly of the pyridone ring and the cyclohexane moiety, as well as their linkage from C-18-C-20. Moreover, four exchangeable protons were observed in the 1 H NMR spectrum (Supplementary Information Table S1), measured in DMSO-d6, which were assigned for NH, 20-OH, 22-OH and 23-OH, respectively. This was supported by HMBC and COSY correlations ( Figure 5) from NH to H-17, 20-OH to C-20, H-22 to 22-OH and H-23 to 23-OH. The above-mentioned analyses accounted for all but one oxygen atom, which was ascribed to the formation of an epoxy bridge between C-19 and C-21, as was also supported by the downfield shifted chemical shift of C-19 (δC 177.2) and upfield shifted chemical shift of C-21 (δC 67.7) compared with those in 2 ( Table 2). The relative configuration of 3 was established via analyses of NOESY data ( Figure 5) and 1 H-1 H couplings ( Table 1). The similarity between the 1 H NMR spectra of the decalin moiety of 3 and 9 supported the common relative stereochemistry, as was also confirmed by NOESY correlations.     (9) [14,15]. Analysis of the NMR data (Tables 1 and 2) for 3 revealed the presence of a decalin moiety as indicated by a close resemblance between its NMR data and those of 9, and the main NMR differences between the two co-metabolites observed for the left-side Fragment A. Further inspection of COSY data ( Figure 5) confirmed the existence of the decalin motif and also established the same spin coupling system from H-21-H 2 -25 as that in 9. In addition, the HMBC correlations ( Figure 5) from H-9 (δ H 4.44) to C-13 and C-14 verified the connection between Fragments A and B, while those from H-17 (δ H 7.69) to C-14, C-15, C-18, C-19 and C-20, along with those from H-25b (δ H 1.51) to C-20 and C-21, enabled the assembly of the pyridone ring and the cyclohexane moiety, as well as their linkage from C-18-C-20. Moreover, four exchangeable protons were observed in the 1 H NMR spectrum (Supplementary Information  Table S1), measured in DMSO-d 6 , which were assigned for NH, 20-OH, 22-OH and 23-OH, respectively. This was supported by HMBC and COSY correlations ( Figure 5) from NH to H-17, 20-OH to C-20, H-22 to 22-OH and H-23 to 23-OH. The above-mentioned analyses accounted for all but one oxygen atom, which was ascribed to the formation of an epoxy bridge between C-19 and C-21, as was also supported by the downfield shifted chemical shift of C-19 (δ C 177.2) and upfield shifted chemical shift of C-21 (δ C 67.7) compared with those in 2 ( Table 2). The relative configuration of 3 was established via analyses of NOESY data ( Figure 5) and 1 H-1 H couplings ( Table 1). The similarity between the 1 H NMR spectra of the decalin moiety of 3 and 9 supported the common relative stereochemistry,  [14,15]. Analysis of the NMR data (Tables 1 and 2) for 3 revealed the presence of a decalin moiety as indicated by a close resemblance between its NMR data and those of 9, and the main NMR differences between the two co-metabolites observed for the left-side Fragment A. Further inspection of COSY data ( Figure 5) confirmed the existence of the decalin motif and also established the same spin coupling system from H-21-H2-25 as that in 9. In addition, the HMBC correlations ( Figure 5) from H-9 (δH 4.44) to C-13 and C-14 verified the connection between Fragments A and B, while those from H-17 (δH 7.69) to C-14, C-15, C-18, C-19 and C-20, along with those from H-25b (δH 1.51) to C-20 and C-21, enabled the assembly of the pyridone ring and the cyclohexane moiety, as well as their linkage from C-18-C-20. Moreover, four exchangeable protons were observed in the 1 H NMR spectrum (Supplementary Information Table S1), measured in DMSO-d6, which were assigned for NH, 20-OH, 22-OH and 23-OH, respectively. This was supported by HMBC and COSY correlations ( Figure 5) from NH to H-17, 20-OH to C-20, H-22 to 22-OH and H-23 to 23-OH. The above-mentioned analyses accounted for all but one oxygen atom, which was ascribed to the formation of an epoxy bridge between C-19 and C-21, as was also supported by the downfield shifted chemical shift of C-19 (δC 177.2) and upfield shifted chemical shift of C-21 (δC 67.7) compared with those in 2 ( Table 2). The relative configuration of 3 was established via analyses of NOESY data ( Figure 5) and 1 H-1 H couplings ( Table 1). The similarity between the 1 H NMR spectra of the decalin moiety of 3 and 9 supported the common relative stereochemistry, as was also confirmed by NOESY correlations.     (Tables 1 and 2) for 4 confirmed this hypothesis, with diagnostic signals for two quaternary sp 2 carbons (δ C 116.6 and 156.2) in 4 instead of the oxyquaternary sp 3 carbons at δ C 76.9 (C-20) and δ C 67.7 (C-21) in 3. This observation led to the suggestion that the furan ring is formed between the cyclohexadiol and 2-piperidone rings and extended the conjugation system of the pyridone chromophore, which was further confirmed by the UV data of 4 with an absorption peak red-shifted to 367 nm. Further examination of COSY and HMBC correlations ( Supplementary Information Figures S42 and S43) corroborated the aforementioned structural assignment. The similarity of the NMR data of the decalin moieties of 4 and 3 suggested the common relative configurations as also confirmed by NOESY correlations (Supplementary Information Figure S44). The crucial NOESY correlation from H-23-H-25b and proton coupling constants of J 22 , 23 (3.8 Hz) and J 23 , 24a (10.8 Hz) defined the relative configurations at C-22 and C-23 as shown in Figure 1.  (Tables 2 and 3) for 5 revealed high similarity to those of apiosporamide (9) [14,15], and the major differences were attributable to the shielded signals for the oxirane ring in 9 instead of two oxymethines (δ H 3.87 and 3.35; δ C 78.7 and 86.6) and a methoxy group (δ H 3.63; δ C 60.5) in 5. Figures S50 and S51) corroborated the aforementioned observations with a key HMBC correlation from the methoxy protons to C-22 (δ C 86.6) and established the planar structure of 5. The relative stereochemistry of 5 was determined to be identical to that of 2 based on the comparison of their NMR spectra, including crucial 1 H-1 H couplings between the corresponding chiral centers, which were further confirmed by examination of the NOESY spectrum (Supplementary Information Figure S52). Analysis of the NMR data (Tables 2 and 3) for 6 suggested that it was a methoxy regio-isomer of 5, and the methoxy group was determined to locate at C-20 (δ C 82.4) via the HMBC correlation from the methoxy protons (δ H 3.18) to C-20. Detailed inspection of COSY and HMBC spectra ( Supplementary  Information Figures S57 and S58) verified the planar structure of 6, and the relative configuration was  (9) [14,15]. Analysis of the NMR data (Tables 2 and 3) for 7 confirmed this hypothesis with diagnostic signals for an oxymethylene (δ H 3.39, 2H; δ C 68.6) in 7 instead of the CH 3 -11 group in 9. Good resemblance between the remaining NMR data for 7 and 9 suggested the assignment of common structural features between the two co-metabolites and indicated their same relative stereochemistry. The structure with the relative configuration of 7 was finally confirmed by further examination of 2D NMR data, especially COSY, HMBC and NOESY spectra ( Supplementary Information Figures S64-S66). Similarly, the oxidation of 8 occurred at C-4 as supported by the COSY correlations from H-4 (δ H 2.73) to H-3 (δ H 1.39) and H-5 (δ H 1.77), as well as the HMBC correlation from H 3 -11 (δ H 1.03) to C-4 (δ C 79.6). The coupling constants of H-4 (dd, J = 9.9 Hz) indicated that it was axially located, and thus, the hydroxyl group was equatorially oriented. The low chemical shift of H-4 (<3.0 ppm) was due to its axial position, which located it in the shielding zone of C2-C3 and C5-C6 bonds. The similarity between the remaining NMR data for 8 and 7, especially key 1 H-1 H coupling constants, supported common structural features, including configurations of the stereocenters, between the two co-metabolites. The structure of 8 with the relative configuration was further confirmed by analysis of 2D NMR data ( Supplementary Information Figures S71-S73). Compounds 9 and 10 were identified to be the previously reported apiosporamide [14,15] and arthpyrone B [4], respectively, based on comprehensive spectroscopic analyses including MS, NMR, [α] D and ECD. While the absolute configuration of 1 was established by X-ray analysis and chemical transformation, those of the other analogues were assigned mainly by analysis of their ECD data. The absolute configurations of 3 and 5-8, which have the same 3-acyl pyridone chromophore, were characterized by their ECD spectra, which are similar to that of 9 (Figure 6), whose absolute structure had been confirmed by total synthesis of its enantiomer [14,15]. The absolute configurations of the stereocenters of 2 and 4 were proposed on biogenetic grounds (Scheme 1). characterized by their ECD spectra, which are similar to that of 9 ( Figure 6), whose absolute structure had been confirmed by total synthesis of its enantiomer [14,15]. The absolute configurations of the stereocenters of 2 and 4 were proposed on biogenetic grounds (Scheme 1). With all these metabolites in hand, we were able to propose a plausible biosynthetic pathway for the new analogues. Compound 1 could be obtained from 10 via one-step Baeyer-Villiger oxidation, while selective oxidation of 9 at C-11 and C-4 would afford 7 and 8, respectively. The likely biosynthetic origin of 2-6 is outlined in Scheme 1. Protonation of the epoxy group of 9 would give the intermediate (i) that could undergo intramolecular nucleophilic substitution by 19-OH, followed by deprotonation to yield 3. Subsequent dehydration of 3 would furnish 4. The intermediate (i) could also experience ring opening by hydrolysis followed by deprotonation to afford the intermediate (ii), which was not obtained in the current study. The intermediate (ii) could produce 2 via Baeyer-Villiger oxidation or undergo selective methylation to yield 5 or 6. Scheme 1. Proposed biosynthetic pathway for 2-6.

Biological Activity
Compounds 1-10 were tested against two Gram-positive strains Mycobacterium smegmatis ATCC 607 and Staphylococcus aureus ATCC 25923, two Gram-negative strains Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027 and a yeast Candida albicans ATCC10231. Compounds 3-6 and 9 exhibited antibacterial activity against M. smegmatis and S. aureus with IC50 values ranging from 1.66-42.8 μM (Table 4), while none of the tested compounds showed significant antimicrobial With all these metabolites in hand, we were able to propose a plausible biosynthetic pathway for the new analogues. Compound 1 could be obtained from 10 via one-step Baeyer-Villiger oxidation, while selective oxidation of 9 at C-11 and C-4 would afford 7 and 8, respectively. The likely biosynthetic origin of 2-6 is outlined in Scheme 1. Protonation of the epoxy group of 9 would give the intermediate (i) that could undergo intramolecular nucleophilic substitution by 19-OH, followed by deprotonation to yield 3. Subsequent dehydration of 3 would furnish 4. The intermediate (i) could also experience ring opening by hydrolysis followed by deprotonation to afford the intermediate (ii), which was not obtained in the current study. The intermediate (ii) could produce 2 via Baeyer-Villiger oxidation or undergo selective methylation to yield 5 or 6. characterized by their ECD spectra, which are similar to that of 9 (Figure 6), whose absolute structure had been confirmed by total synthesis of its enantiomer [14,15]. The absolute configurations of the stereocenters of 2 and 4 were proposed on biogenetic grounds (Scheme 1). With all these metabolites in hand, we were able to propose a plausible biosynthetic pathway for the new analogues. Compound 1 could be obtained from 10 via one-step Baeyer-Villiger oxidation, while selective oxidation of 9 at C-11 and C-4 would afford 7 and 8, respectively. The likely biosynthetic origin of 2-6 is outlined in Scheme 1. Protonation of the epoxy group of 9 would give the intermediate (i) that could undergo intramolecular nucleophilic substitution by 19-OH, followed by deprotonation to yield 3. Subsequent dehydration of 3 would furnish 4. The intermediate (i) could also experience ring opening by hydrolysis followed by deprotonation to afford the intermediate (ii), which was not obtained in the current study. The intermediate (ii) could produce 2 via Baeyer-Villiger oxidation or undergo selective methylation to yield 5 or 6. Scheme 1. Proposed biosynthetic pathway for 2-6.

Biological Activity
Compounds 1-10 were tested against two Gram-positive strains Mycobacterium smegmatis ATCC 607 and Staphylococcus aureus ATCC 25923, two Gram-negative strains Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027 and a yeast Candida albicans ATCC10231. Compounds 3-6 and 9 exhibited antibacterial activity against M. smegmatis and S. aureus with IC50 values ranging from 1.66-42.8 μM (Table 4), while none of the tested compounds showed significant antimicrobial Scheme 1. Proposed biosynthetic pathway for 2-6.

Biological Activity
Compounds 1-10 were tested against two Gram-positive strains Mycobacterium smegmatis ATCC 607 and Staphylococcus aureus ATCC 25923, two Gram-negative strains Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027 and a yeast Candida albicans ATCC10231. Compounds 3-6 and 9 exhibited antibacterial activity against M. smegmatis and S. aureus with IC 50 values ranging from 1.66-42.8 µM (Table 4), while none of the tested compounds showed significant antimicrobial activity Mar. Drugs 2018, 16, 174 9 of 14 against E. coli, P. aeruginosa and C. albicans at 50 µM. Expanding our biological activity evaluations, these isolates were also screened for AChE inhibitory and cytotoxic activities against two human osteosarcoma cell lines U2OS and MG63. Only 9 displayed mild cytotoxicity against the two cell lines with IC 50 values of 19.3 and 11.7 µM.

Fungal Material
The fungus strain UJNMF0008 was isolated from a marine sediment sample collected in the South China Sea (17 • 55 00 N, 115 • 55 31 E; 3858 m depth). This strain was identified as an Arthrinium sp. based on morphological traits and a molecular biological protocol by DNA amplification and comparison of its ITS region sequence with the GenBank database (100% similarity with Arthrinium sp. zzz1842 (HQ696050.1)). The BLAST sequenced data were deposited at GenBank (No. MG010382). The strain was deposited at China General Microbiological Culture Collection Center (CGMCCC), Institute of Microbiology, Chinese Academy of Sciences.

Fermentation and Extraction
Arthrinium sp. UJNMF0008 from a PDA culture plate was inoculated in 500-mL Erlenmeyer flasks containing 150 mL soluble starch medium (1% glucose, 0.1% soluble starch, 1% MgSO 4 , 0.1% KH 2 PO 4 , 0.1% peptone and 3% sea salt) at 28 • C on a rotary shaker at 180 rpm for 3 days as seed cultures. Then, each of the seed cultures (20 mL) was transferred into autoclaved 1-L Erlenmeyer flasks with solid rice medium (each flasks contained 80 g commercially available rice, 0.4 g yeast extract, 0.4 g glucose, and 120 mL water with 3% sea salt). After that, the strain was incubated statically for 30 days at 28 • C.
After fermentation, the total 4.8 kg rice culture was crushed and extracted with 15.0 L 95% EtOH three times. The EtOH extract was evaporated under reduced pressure to afford an aqueous solution and then extracted with 2.0 L ethyl acetate three times to give 80 g crude gum.  Tables 2 and 3

Antimicrobial Assays
The antimicrobial activity was assayed against the Gram-positive bacterial strains Mycobacterium smegmatis ATCC 607 and Staphylococcus aureus ATCC 25923, Gram-negative Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027 and yeast Candida albicans ATCC10231 by liquid growth inhibition in 96-well microplates. Briefly, precultures of the tested microorganisms were made by inoculating 10 mL of medium (LB medium for bacteria and YM medium for fungus) and incubated for 24 h at 37 • C for bacteria or 48 h at 28 • C for fungus. Then, the cell density was adjusted to 10 4 −10 5 cfu mL −1 by the corresponding broth. An aliquot of 200 µL of the microbial suspension was distributed in each well containing 2-fold serial dilution of the tested compounds. The plate was incubated at 37 • C for 12 h for the bacteria or at 28 • C for 48 h for the fungus, and the optical density of each well was measured at 600 nm spectrophotometrically. IC 50 values were defined as the concentration of a compound resulting in a 50% decrease in the number of microbial cultures compared to the blank control. The experiment was run in three replicates.

AChE Inhibitory Assay
The AChE inhibitory activities of compounds were tested by using the modified Ellman's method as described previously [19].

Cytotoxic Assay
The cytotoxicity of compounds was evaluated using human osteosarcoma U2OS and MG63 cell lines by the MTT method as described previously [20]. Adriamycin was used as a positive control (IC 50 21.1 and 3.56 nM for U2OS and MG63, respectively).
3.9. X-ray Diffraction Analysis of Arthpyrone D (1) Compound 1 was crystallized in MeOH-H 2 O at room temperature. The X-ray crystallographic data were obtained from an Oxford Gemini E Eos CCD detector equipped with a graphite monochromated Cu Kα radiation (λ = 1.54178 Å) at 293(2) K. The structure was solved with the XT structure solution program using intrinsic phasing and refined with the ShelXL refinement package using least squares minimization [21,22]. All non-hydrogen atoms were refined anisotropically. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. Crystallographic data for 1 were deposited at the Cambridge Crystallographic Data Centre (Deposition No.: CCDC 1813453).

Conclusions
Until now, about twenty 4-hydroxy-2-pyridone alkaloids with a decalin ring have been reported and showed significant cytotoxicity, antimicrobial activity and AChE inhibitory activity, but only arthpyrones A-C were isolated from marine-derived fungus [6,14,15,[23][24][25][26][27][28][29][30]. Our current study contributed eight new members (1)(2)(3)(4)(5)(6)(7)(8) to this structural class from a deep-sea-derived fungus Arthrinium sp. Among this series of fungal metabolites, the ester bridge that connected the pyridone and decalin fragments in 1 and 2 was reported for the first time, while 3 and 4 incorporating the rare hexa-or tetrahydrobenzofuro[3,2-c]pyridin-3(2H)-one ring system were also the first examples of this class of compounds. Our biological activity evaluations established selective compounds as antibacterial agents against Gram-positive M. smegmatis and S. aureus and/or cytotoxic agents toward human osteosarcoma cell lines U2OS and MG63. Knowledge of these rare pyridone alkaloids will be of great interest to the scientific community.