Antibacterial Polyketides Isolated from the Marine-Derived Fungus Fusarium solani 8388

Seven new polyketides named fusarisolins F-K (1–6) and fusarin I (7) were isolated from the marine-derived fungus Fusarium solani 8388, together with the known anhydrojavanicin (8), 5-deoxybostry coidin (9), and scytalol A (10). Their structures were established by comprehensive spectroscopic data analyses, and by comparison of the 1H and 13C NMR data with those reported in literature. Fusarisolin F (1) contained both a dichlorobenzene group and an ethylene oxide unit, which was rare in nature. In the bioassays, fusarisolin I (4), fusarisolin J (5), and 5-deoxybostry coidin (9) exhibited obvious antibacterial activities against methicillin-resistant Staphylococcus aureus n315 with MIC values of 3, 3, and 6 μg/mL, respectively. Fusarisolin H (3) and fusarisolin J (5) showed inhibitory effects against methicillin-resistant Staphylococcus aureus NCTC 10442 with the same MIC value of 6 μg/mL. With the exception of 5, all other compounds did not show or showed weak cytotoxicities against HeLa, A549, and KB cells; while fusarisolin J (5) demonstrated moderate cytotoxicities against the three human cancer cell lines with CC50 values between 9.21 and 14.02 μM.


Introduction
The deep sea is a special environment with high pressure, high salt, low temperature, low oxygen concentration, darkness, and oligotrophic conditions. The microorganisms that inhabit the deep sea are usually obviously different with those living on the mainland. The severe growth environment allows microorganisms to produce and accumulate various secondary metabolites with novel chemical structures and potent physiological activities. Therefore, marine microorganisms have been considered as a reservoir of bioactive secondary metabolites [1,2]. Fusarium species are ubiquitous in both marine and terrestrial environments, including deserts and the Arctic [3], which produce mycotoxins as deoxynivalenol, zearalenone, fumonisin B1, and T-2 toxin which causes the risks of bakanae, foot rot, scab, and head blight [4,5]. In addition, Fusarium species possess the potential capability to produce structurally diverse secondary metabolites such as polyketides, alkaloids, terpenoids, peptides, and steroids with potent physiological activities [3,6]. Polyketides are a class of natural secondary metabolites synthesized by polyketide synthases, which have diverse frameworks and outstanding pharmacological activities. Some marine-derived polyketide compounds have already played important roles for the development of new drugs [7][8][9]. For example, the polyketide salinosporamide A, isolated from a marine actinomycete, is a potent proteasome inhibitor and is now in clinical trials for the treatment of brain cancer [10]. Abyssomicin C, a polyketide from marine actinomycetes, demonstrates significant antibacterial activity against the methicillin-and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) strains by inhibiting formation of p-aminobenzoate [11,12].
Compound 3 was isolated as a red powder. The (−)HRESIMS spectrum showed signal at m/z 303.0877 [M − H] − (calcd. for C 16 H 15 O 6 − , 303.0874), established the molecular formula of C 16 H 16 O 6 for 3 with the aid of 13 C NMR data, inferring 9 degrees of unsaturation. The 1 H and 13 C NMR spectroscopic data of 3 (Table 2) showed resonances consistent with a hydrogen-bonded phenol moiety at δ H 12.45 (br s, OH-6), a methyl (δ H 1.52, δ C 22.9, Me-3), two methoxyls (δ H 3.98, δ C 49.0, OMe-3, δ H 3.29, δ C 56.5, OMe-7), two methylenes (CH 2 -1; CH 2 -4), two aromatic methines (δ H 7.05, δ C 115.0, CH-8; δ H 7.63, δ C 120.6, CH-9), six non-protonated aromatic carbons, two carbonyl carbon at δ C 181.6 (C-10) and δ C 189.4 (C-5), and a ketal or a hemiketal carbon at δ C 97.2 (C-3). The comparison of these NMR data with those reported for 3-methyl ether fusarubin showed close similarity [14], inferring a pyranonaphthoquinone framework for 1. The HMBC correlations originated from H 2 -1, H 2 -4, H-8, H-9, and the hydrogen atom of OH-6 confirmed the elucidation of pyranonaphthoquinone core (Figure 4). The location of OMe-3, Me-3, and OMe-7 were determined by the HMBC correlations of OMe-3/C-3, Me-3/C-3, and OMe-7/C-7, respectively. Thus, the planar structure of 3 was established. The absolute configuration of the stereogenic center at C-3 was assigned as S on the basis of the ECD curve of 3 showed good agreement of the calculated one for 3S-3 ( Figure 5A). Compound 3 was named fusarisolin H.         Table 2), except that the additional signals at δ H 2.65 and δ C 23.4 (Me-9) attributable to a methyl group were observed in 4. Moreover, the aromatic proton at δ H 7.63 (H-9) in 3 had disappeared in 4. In addition, the 13 C NMR signal of C-9 was shifted downfield from δ C 120.6 in 3 to 136.6 in 4. These changes indicated that H-9 in 3 was replaced a methyl group in 4. In the HMBC spectrum, the correlation from the new appearing methyl protons to C-8, C-9, and C-9a confirmed the presence of the Me-9. The ECD spectrum of 4 showed a negative Cotton effect at 260 nm and positive Cotton effect at 293 nm ( Figure 5B), which were contrary to those of 3, establishing 3R configuration for 4. Compound 4 was named fusarisolin I.  32 (H 3 -3 ). The 13 C NMR spectrum revealed signals corresponding to two carbonyls at δ C 190.8 (C-4) and 184.5 (C-1), three aromatic methine carbons, and five non-protonated aromatic carbons, which were attributable to a naphthoquinone scaffold [15]. In addition, three aliphatic carbon signals, including an oxygen-bearing methine carbon at δ C 67.9 (C-2 ), a methylene carbon at δ C 36.2 (C-1 ), and a methyl carbon at δ C 24.4 (C-3 ) were observed. The COSY correlations of H-6/H-7/H-8, together with the HMBC correlations from H-6 to C-8, from H-7 to C-5, C-8a, from H-8 to C-6, C-4a, C-1, from 5-OH to C-4a, C-5, C-6, and from Me-2 to C-1, C-2, C-3 established a 2,3-disubstituted-5-hydroxy naphthoquinone skeleton. In addition, the COSY correlations of H 2 -1 /H-2 /H 3 -3 confirmed the presence of the side chain of CH 2 -1 /CH-2 /CH 3 -3 . The HMBC correlations of H 2 -1 to C-2, C-3, C-4 revealed the location of the side chain at C-3. The absolute configuration of the stereogenic center at C-2 was determined by calculation of ECD spectra. The experimental ECD spectrum of 5 was in good agreement with that of (2 S)-5 ( Figure 6), establishing S configuration for C-2 . Compound 5 was elucidated as (S)-5-hydroxy-3-(2-hydroxypropyl)-2-methyl naphthalene-1,4-dione and given the name fusarisolin J.     (Table 4) revealed the presence of four methyls (Me-1 , Me-8 , Me-9 , Me-10 ), one methoxyl (OMe-4), two methylene (CH 2 -4 and CH 2 -6 ), three aromatic (CH-3, CH-5, CH-3 ) and two aliphatic methines (CH-4 and CH-6 ), three non-protonated carbons (C-4, C-6, C-2 ), and a carbonyl (C-2). The COSY spectrum established a long alkyl chain of C-3 ~C-8 with substitutions of two methyl groups at C-4 and C-6 ( Figure 7). The HMBC correlations from the protons of Me-1 to C-2 and C-3 , as well as the 1 H NMR chemical shift of Me-1 at δ H 1.85, confirmed the structure of the entire side chain (C-1 ~C-8 ). Further HMBC correlations from H-3 to C-4, and C-5, and from H-5 to C-3, C-4, and C-6 established a buta-1,3-diene fragment (C-3~C-6). The 13 C NMR chemical shift values of C-4 at δ C 171.7 and C-6 at δ C 161.5 indicated C-4 and C-6 were oxygen-bearing carbons. The remaining one degree of unsaturation suggested the presence of a ring in 6. Importantly, the HMBC correlation from H-3 to the carbonyl (C-2) was observed. The carbonyl was connected to C-6 through an ester bond, constructing a 2H-pyran-2-one scaffold. The HMBC correlation of OMe-4/C-4 validated the position of OMe-4. The HMBC correlations from H-5 to C-2 and from H 3 -1 and H-3 to C-6 confirmed the linkage of C-6/C-2 , inferring the position of the side chain at C-6.  In the NOESY spectrum, the correlation between H-3′ and H3-1′ was observed, confirming the E configuration of the double bond between C-2′ and C-3′. The absolute configurations of the stereogenic centers at C-4′ and C-6′ in 6 were determined by ECD calculation. Based on the experimental ECD spectrum of 6 being consistent with that of 4′S,6′S-6 ( Figure 8A), the configurations of 6 were assigned as 4′S, 6′S. Compound 6 was In the NOESY spectrum, the correlation between H-3 and H 3 -1 was observed, confirming the E configuration of the double bond between C-2 and C-3 . The absolute configurations of the stereogenic centers at C-4 and C-6 in 6 were determined by ECD calculation. Based on the experimental ECD spectrum of 6 being consistent with that of 4 S,6 S-6 ( Figure 8A), the configurations of 6 were assigned as 4 S, 6 S. Compound 6 was designated as fusarisolin K. In the NOESY spectrum, the correlation between H-3′ and H3-1′ was observed, confirming the E configuration of the double bond between C-2′ and C-3′. The absolute configurations of the stereogenic centers at C-4′ and C-6′ in 6 were determined by ECD calculation. Based on the experimental ECD spectrum of 6 being consistent with that of 4′S,6′S-6 ( Figure 8A), the configurations of 6 were assigned as 4′S, 6′S. Compound 6 was designated as fusarisolin K. Compound 7 was isolated as yellow oil. It showed a sodium adducted ion peak at m/z 301.1411 [M + Na] + (calcd. for C16H22NaO4 + , 301.1410) in the (+)HRESIMS spectrum. The molecular formula of 7 was established to be C16H22O4, indicating 6 degrees of unsaturation. The 1 H and 13 C NMR spectroscopic data of 7 (Table 4) were characterized by chemical resonances consistent with three methyls (Me-1, Me-12, Me-15), two methoxyls (OMe-14, OMe-16), six aromatic and one aliphatic methines, two non-protonated carbons, and one keto and one esteric carbonyls (C-11, C-13). These data were similar with those for fusarin J [16], except one more signal attributed for a methoxyl at δH 3.33 and δC 56.3 (OMe-16) was observed in 7. Detailed analyses of the HMBC correlations for 7 (Figure 7) placed the new-appearing methoxyl at C-4 and confirmed the structure of 7. Compound 7 showed a positive specific rotation value, which was the same as that of fusarin J, indicating the 4S configuration. In addition, the experimental ECD spectrum of 7 was coincidental with the calculated one of 4S-7 ( Figure 8B), confirmed the determination of 4S configuration. Compound 7 was named fusarin I.
In addition, these isolates were tested for cytotoxicities against human lung adenocarcinoma cell line A549, human cervical carcinoma cell line HeLa, and human nasopharyngeal carcinoma cell line KB using MTT colorimetric assays. Fusarisolin J (5) inhibited cell proliferation of HeLa, A549, and KB with CC 50 values of 9.21, 14.02, and 12.07 µM, respectively. With the exception of 5, other compounds did not show or showed weak cytotoxicities against the three human cancer cell lines (Table 6).

Fungal Identification, Fermentation, and Extract
The fungus Fusarium solani 8388 was isolated from sediments collected in the Shenhu area of the South China Sea at a depth of 100 m. First, 1 g of sediment was suspended in 100 mL of autoclaved sea water. Then 1 mL of suspension was diluted with 100 mL of autoclaved sea water. After that, 1 mL of dilution was added into 20 mL of PDA medium, which containing chloramphenicol at a concentration of 100 mg/L. The PDA medium plate was cultured at 28 • C for 5 days. The cultured mycelia were purified to obtain single colony of strain 8388 using PDA medium. The internal transcribed spacer (ITS) region was amplified and sequenced using the general primers ITS1 and ITS4. The ITS region of the fungus was a 572-bp DNA sequence (GenBank accession number: KT336512), which showed 99.07% identity to Fusarium solani. The cladogram is shown in Figure S8 in Supplementary Materials. The strain was deposited at the School of Pharmacy, Guangzhou Medical University.
The producing strain was incubated on a potato dextrose agar medium plate under 28 • C for 3 days. Then, the fresh mycelia were inoculated to a 250 mL Erlenmeyer flask containing 0.2 g of peptone, 0.1g of yeast extract, 1.0 g of glucose, and 100 mL of seawater. The flasks were incubated on a rotating shaker at 28 • C for 7 days to produce mycelia. The mycelia were inoculated to 1 L Erlenmeyer flasks containing 100 g of rice, 0.5 g of yeast extract, 0.5 g of glucose, 3 g of crude salt, and 200 mL of water. In total, 200 flasks were used. The flasks were incubated statically at 28 • C for 32 days. The fermented cultures were extracted with MeOH three times. After evaporation under reduced pressure, the extract was re-dissolved in water and extracted with EtOAc (1:1) three times. The EtOAc layer was evaporated under reduced pressure to obtain 320 g of extract.

X-ray Diffraction
Colorless crystals of 1 were obtained from MeOH by slow evaporation. The crystal data were collected on an Agilent Gemini Ultra diffractometer with Cu Kα radiation (λ = 1.54184 Å) at 170.00(10) K. The crystal structure was solved with the SHELXT structure solution program using Intrinsic Phasing and refined with the SHELXL refinement package using Least Squares minimisation [19,20].
Crystal Crystal data and structure refinements for 1 are listed in Tables S1-1-S1-7 in Supplementary Materials.

Calculation of ECD
MOE 2019 software was used for conformational search. The geometries of all conformers for ECD calculations were optimized sequentially using Gaussian 09W software at RHF/6-31G(d,p) level. The TDDFT method was employed for the ECD calculations of these compounds at the RB3LYP/6-31G (d,p) level in methanol [21].

Antibacterial Assays
The antibacterial activities of compounds 1-10 were assessed against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, the methicillin-resistant strains Staphylococcus aureus NCTC 10442 and Staphylococcus aureus n315 using a sequential 2-fold serial dilution method, in which compounds were tested at final concentrations ranging from 100 to 0.7 µg/mL. Compounds were dissolved in DMSO, serially diluted in Mueller-Hinton (M-H) broth. The test was conducted in triplicate using 96-well plates; each well contained 200 µL of liquid. Amoxicillin and vancomycin were used as positive controls. DMSO in M-H broth was used as blank control [22].

Cytotoxic Assays
The cytotoxic activities of compounds 1-10 were evaluated using the MTT colorimetric assay against A549, HeLa, and KB human tumor cells using the previously reported MTT method [23]. Briefly, human tumor cells were seeded in 96-well plates at a density of 2.5 × 10 4 cells/mL and incubated at 37 • C in a humidified incubator (5% CO 2 ) for 24 h. After that, various concentrations of compounds were added and incubated for 48 h. Then, 20 µL of MTT solution (5 mg/mL) was added to each well, and the cells were further incubated for 4 h. The culture supernatant was removed, and 100 µL of DMSO was added to dissolve the MTT-formazan crystals. Cell growth inhibition was measured by recording the absorbance at λ = 540 nm using a microplate reader and calculated using the following equation: growth inhibition = (1 − OD of treated cells/OD of control cells) × 100%. The half maximal inhibitory concentration (CC 50 ) values were obtained from the concentrationresponse curves, which were plotted for each tested compound using software GraphPad Prism 9.0. The results were expressed as the mean value of triplicate data points.

Conclusions
In this study, the secondary metabolites of fungus Fusarium solani 8388 isolated from the Shenhu area in the South China Sea were investigated. Seven new polyketide compounds named fusarisolins F-K (1-6) and fusarin I (7), as well as three known analogues (8)(9)(10) were isolated and identified. Fusarisolin I (1) comprised both a dichlorobenzene group and an ethylene oxide unit, which was rare in nature. In the in vitro antibacterial bioassays, fusarisolin I (4), fusarisolin J (5), and 5-deoxybostrycoidin (9) exhibited obvious antibacterial activities against methicillin-resistant Staphylococcus aureus n315. Fusarisolin H (3) and fusarisolin J (5) showed inhibitory effects against methicillin-resistant Staphylococcus aureus NCTC 10442. With the exception of 5, all other compounds did not show or showed weak cytotoxicities against human HeLa, A549, and KB cells.