Antiviral Cyclopropane Acids from Deep-Sea-Derived Fungus Aspergillus sydowii

Four novel monocyclic cyclopropane acids, namely, sydocyclopropanes A–D (1–4), along with one known congener hamavellone B (5), were isolated from the Aspergillus sydowii MCCC 3A00324 fungus, which was isolated from the deep-sea sediment. The gross structures of novel compounds were established by detailed analyses of the spectroscopic data (HRESIMS and NMR spectra), and their absolute configurations were resolved on the basis of the quantum chemical calculations of ECD and NMR data, in association with DP4+ probability analyses. Sydocyclopropanes A–D, featuring the 1,1,2,3-tetrasubstituted cyclopropane nucleus with different lengthy alkyl side chains, were discovered in nature for the first time. All compounds exhibited antiviral activities against A/WSN/33 (H1N1), with IC50 values ranging from 26.7 to 77.2 μM, of which compound 1 exhibited a moderate inhibitory effect (IC50 = 26.7 μM).

our continuous efforts to find more new or active compounds, subsequent chromatography of the minor components of the fractions of the EtOAc extract of the fungus obtained four novel cyclopropane acids (sydocyclopropanes A-D, 1-4) and one known congener hamavellone B (5) (Figure 1). Compounds 1-4 are the first representatives of single ring nucleus featuring 1,1,2,3-tetrasubstituted cyclopropane ring with different carbon side chains. All the isolated compounds were evaluated their antiviral effects against A/WSN/33 (H1N1), and 1-5 exhibited inhibitory effects with IC50 values ranging from 26.7 to 77.2 μM. Herein, the isolation, structural identification, and anti-H1N1 activities of 1-5 are presented.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 2 of 10 our continuous efforts to find more new or active compounds, subsequent chromatography of the minor components of the fractions of the EtOAc extract of the fungus obtained four novel cyclopropane acids (sydocyclopropanes A-D, 1-4) and one known congener hamavellone B (5) (Figure 1). Compounds 1-4 are the first representatives of single ring nucleus featuring 1,1,2,3-tetrasubstituted cyclopropane ring with different carbon side chains. All the isolated compounds were evaluated their antiviral effects against A/WSN/33 (H1N1), and 1-5 exhibited inhibitory effects with IC50 values ranging from 26.7 to 77.2 μM. Herein, the isolation, structural identification, and anti-H1N1 activities of 1-5 are presented.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 6 of 10 structure to be identical to that of 3. The large 3 JH2,H-3 value (9.5 Hz) between H-2 and H-3, indicating the cis orientation of the vicinal protons, in association with the NOESY correlations from H2-4 (δH 1.59) to H3-8 (δH 1.03) and H3-9 (δH 1.15) and from H3-8 to H3-9 deduced its relative configurations to be 1S*, 2R*, and 3S* (Figure 3). In addition, the experimental ECD data of 4 matched well with the calculated ECD data of (1S,2R,3S)-4, indicating the S configurations for C-1 and C-3, and R for C-2 ( Figure 7). Therefore, the structure of 4 was assigned as a C-2 epimer of 3 and named sydocyclopropane D.  Compound 4 has the same molecular formula as that of 3, as determined by the HRESIMS (m/z 207.1002, [M + Na] + ) and 13 C NMR spectra. The 1 H and 13 C NMR data were nearly identical to those of 3, revealing a structurally similar analogue. The differences were attributed to the shielded chemical shifts of H-2 (∆δ H −0.72) and the deshielded H 3 -8 (∆δ H 0.13), C-1 (∆δ C 3.7), C-2 (∆δ C 7.2), C-8 (∆δ C 5.2), and C-9 (∆δ C 7.0) when compared with the corresponding NMR data of 3. Analysis of the 2D NMR spectra of 4 established its gross structure to be identical to that of 3. The large 3 J H2,H-3 value (9.5 Hz) between H-2 and H-3, indicating the cis orientation of the vicinal protons, in association with the NOESY correlations from H 2 -4 (δ H 1.59) to H 3 -8 (δ H 1.03) and H 3 -9 (δ H 1.15) and from H 3 -8 to H 3 -9 deduced its relative configurations to be 1S*, 2R*, and 3S* (Figure 3). In addition, the experimental ECD data of 4 matched well with the calculated ECD data of (1S,2R,3S)-4, indicating the S configurations for C-1 and C-3, and R for C-2 ( Figure 7). Therefore, the structure of 4 was assigned as a C-2 epimer of 3 and named sydocyclopropane D.
tions from H2-4 (δH 1.59) to H3-8 (δH 1.03) and H3-9 (δH 1.15) and from H3-8 to H3-9 deduced its relative configurations to be 1S*, 2R*, and 3S* (Figure 3). In addition, the experimental ECD data of 4 matched well with the calculated ECD data of (1S,2R,3S)-4, indicating the S configurations for C-1 and C-3, and R for C-2 ( Figure 7). Therefore, the structure of 4 was assigned as a C-2 epimer of 3 and named sydocyclopropane D.  Apart from compounds 1-4, one known metabolite was obtained and established to be hamavellone B (5) on the basis of the comparison of the NMR data with those reported in the literature [11,29].
All the isolated compounds were evaluated the anti-influenza virus A/WSN/33 (H1N1) activities using the cytopathic effect (CPE) reduction assay [30], and oseltamivir (OSV) was used as the positive control. Cytotoxic evaluation was carried out via CellTiter-Glo assay to determine whether the antivirus effects was due to the toxicity of the tested compounds against MDCK cells. As a result, all the isolated compounds exhibited no cytotoxic activities at the concentration of 100 μM, and compounds 1-5 showed inhibitory H1N1 effects with IC50 values ranging from 26.7 to 77.2 μM (Table 3). Preliminary analysis of the structure-activity relationships found that the methyl 2-hydroxy-4-oxobutanoate side chain residing at C-1 in 1 significantly enhanced the antiviral activity, as evidenced by 1 exhibiting an IC50 value of 26.7 μM, while 3 showed 77.2 μM. Additionally, the C-3 chiral center had little effect on anti-H1N1 activities as exemplified by the C-3 epimers of 3 (IC50 = 77.2 μM) and 4 (IC50 = 66.4 μM). Apart from compounds 1-4, one known metabolite was obtained and established to be hamavellone B (5) on the basis of the comparison of the NMR data with those reported in the literature [11,29].
All the isolated compounds were evaluated the anti-influenza virus A/WSN/33 (H1N1) activities using the cytopathic effect (CPE) reduction assay [30], and oseltamivir (OSV) was used as the positive control. Cytotoxic evaluation was carried out via CellTiter-Glo assay to determine whether the antivirus effects was due to the toxicity of the tested compounds against MDCK cells. As a result, all the isolated compounds exhibited no cytotoxic activities at the concentration of 100 µM, and compounds 1-5 showed inhibitory H1N1 effects with IC 50 values ranging from 26.7 to 77.2 µM (Table 3). Preliminary analysis of the structure-activity relationships found that the methyl 2-hydroxy-4-oxobutanoate side chain residing at C-1 in 1 significantly enhanced the antiviral activity, as evidenced by 1 exhibiting an IC 50 value of 26.7 µM, while 3 showed 77.2 µM. Additionally, the C-3 chiral center had little effect on anti-H1N1 activities as exemplified by the C-3 epimers of 3 (IC 50 = 77.2 µM) and 4 (IC 50 = 66.4 µM).

General Experimental Procedures
Optical rotation data were recorded on the basis of the Anton Paar MCP 500 automatic polarimeter. The UV and ECD spectra were measured by the Shimadzu UV-1800 spectrophotometer and chirascan CD spectrometer, respectively. The Bruker Avance-400 FT NMR spectrometer was used to measure the NMR data. Chemical shifts (δ) were referenced to the CD 3 OD at 3.31 and 49.00 ppm for proton and carbon, respectively. The Xevo G2 Q-TOF mass spectrometer was used to record the HRESIMS spectra. Silica gel, sephadex LH-20, and ODS-A were used for column chromatography (CC). All solvents used for CC were analytical grade. Precoated silica gel plates were used for the thin-layer chromatography (TLC) analysis, and the TLC spots were visualized by heating the plates when sprayed with vanillin sulfuric acid chromogenic reagent. Semipreparative HPLC was performed on a Alltech LS class pump equipped with UV/Vis detector using YMC packed ODS-A (250 × 10 mm, 5 µm) column for the purification.

Fungal Strain, Identification, and Fermentation
The

Extraction, Isolation, and Purification
The fermented substrate was extracted three times using ethyl acetate (EtOAc). The combined EtOAc was concentrated under reduced pressure to yield 16.2 g extract. The EtOAc extract was subjected to CC on silica gel vacuum liquid chromatography (VLC), eluting with increasing polarity from CH 2 Cl 2 to MeOH (1:0~0:1) to yield two fractions (A and B). Fraction B (8.5 g) was chromatographed via ODS CC with MeOH/H 2 O gradient elution (30%~100%) to obtain fourteen subfractions (SF1-SF14). Subfraction SF3 (147 mg) was fractionated on the basis of the CC over silica gel, using petroleum ether (PE) and EtOAc isocratic elution (10:1), and then further purified by semipreparative HPLC eluted with 30% MeOH in H 2 O to obtain 2 (8.1 mg

Anti-Influenza Virus H1N1 Assay
The anti-influenza virus test was carried out as previously reported [30], and influenza A/WSN/33 (H1N1) was used in the study. In brief, Madin-Darby canine kidney (MDCK) cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco BRL, Inc., Gaithersburg, MD, USA) and supplemented with 1% fetal bovine serum (FBS) (PAA Laboratories, Linz, Austria) at 37 • C in 5% CO 2 . The MDCK cells were seeded into 96-well plates (1 × 10 5 cells per well) and incubated for 24 h. Then, the cells were infected with influenza virus A/WSN/33 (multiplicity of infection, MOI = 0.1) and suspended in DMEM supplemented with 1% FBS, the test compounds, and 2 mg/mL TPCK-treated trypsin, with a final DMSO concentration of 1% in each well. After 40 h incubation, CellTiter-Glo reagent (Promega Corp., Madison, WI, USA) was added and the plates were then read using a plate reader (Tecan Infinite M2000 PRO™; Tecan Group Ltd., Mannedorf, Switzerland). Positive control was chosen for oseltamivir (OSV). The IC 50 values were obtained using the Sigma Plot Statistical Analysis software as the test compound concentration required inhibiting cytopathic production after post-infection by 50%.

Cytotoxicity Test
The MDCK cells were grown in DMEM, supplemented with 1% FBS under a humidified atmosphere (5% CO 2 ) at 37 • C. Cell suspension was placed into 96 well microtiter plates and incubated for 24 h. Then, the increasing amounts of test compounds were added to each well and further incubated for 40 h. Cytotoxicity was assessed with the CellTiter-Glo assay, as described above.

ECD Calculations of Compounds 1-4
The conformational searches of model structures 1-4 were performed according to the above NMR calculation description. Searched conformers were further optimized at the B3LYP/6-311G(2d,p) level in the gas phase by Gaussian 09 program. The optimized conformers with a Boltzmann population over 1% were chosen for the ECD calculations. The energies, oscillator strengths, and rotational strengths of the first 60 electronic excitations were calculated at the B3LYP/6-311G(2d,p) level in methanol with the conductor-like polarizable continuum model (CPCM). The calculated ECD data of the conformers were combined on the basis of their weighing the Boltzmann distribution rate using SpecDis 1.71 software [31], and Gaussian function band shape sigma was set as 0.3 eV.

Conclusions
In conclusion, chemical examination of the fermented cultures of the deep-sea sedimentderived Aspergillus sydowii MCCC 3A00324 fungus resulted in the isolation of four novel Mar. Drugs 2022, 20, 410 9 of 10 cyclopropane acids, named sydocyclopropanes A-D (1-4), together with one known congener hamavellone B (5). The novel structures were resolved on the basis of spectroscopic analysis (NMR and HRESIMS data) and ECD and NMR calculations, in association with DP4+ probability analyses. Compounds 1-4 are the first representatives of monocyclic ring systems featuring a 1,1,2,3-tetrasubstituted cyclopropane ring bearing different alkyl side chains, revealing deep-sea-derived fungi are new source of structurally novel compounds. All isolated metabolites were evaluated the anti-H1N1 effects by the CPE reduction assay, and 1-5 exhibited inhibitory activities with IC 50 values ranging from 26.7 to 77.2 µM. Among them, compound 1 showed moderately antiviral activity (IC 50 = 26.7 µM). This study is the first to discover single ring cyclopropane acids in marine-derived fungi, and more importantly, sydocyclopropanes exhibiting anti-H1N1 effects are reported here for the first time.