New Butenolides and Cyclopentenones from Saline Soil-Derived Fungus Aspergillus Sclerotiorum

Three new γ-hydroxyl butenolides (1–3), a pair of new enantiomeric spiro-butenolides (4a and 4b), a pair of enantiomeric cyclopentenones (5a new and 5b new natural), and six known compounds (6–11), were isolated from Aspergillus sclerotiorum. Their structures were established by spectroscopic data and electronic circular dichroism (ECD) spectra. Two pairs of enantiomers [(+)/(–)-6c and (+)/(–)-6d] obtained from the reaction of 6 with acetyl chloride (AcCl) confirmed that 6 was a mixture of two pairs of enantiomers. In addition, the X-ray data confirmed that 7 was also a racemate. The new metabolites (1−5) were evaluated for their inhibitory activity against cancer and non-cancer cell lines. As a result, compound 1 exhibited moderate cytotoxicity to HL60 and A549 with IC50 values of 6.5 and 8.9 µM, respectively, and weak potency to HL-7702 with IC50 values of 17.6 µM. Furthermore, compounds 1−9 were screened for their antimicrobial activity using the micro-broth dilution method. MIC values of 200 μg/mL were obtained for compounds 2 and 3 towards Staphylococcus aureus and Escherichia coli, while compound 8 exhibited a MIC of 50 μ/mL towards Candida albicans.

The Yellow River Delta is formed mainly by the deposition of sand and mud carried by the Yellow River, which is the world's youngest wetland ecosystem [12]. High evaporation and tidal intrusion heavily salinizes and alkalizes the soil, half of which is barren and not suitable for the growth of crops [13,14]. The saline soil is typically characterized by poor nutrient and high salinity, which endows the microorganism special biosynthetic pathways during the evolutionary process to produce structurally novel and biologically active secondary metabolites. Up to now, only a few research papers have been carried out on the fungi from this unique environment [15]. In our continuation of investigation on the saline soil-derived fungi from the Yellow River Delta, dozens of natural products (NPs) with multifarious structural features and a wide range of biological activities
Compound 2 was also isolated as an optically inactive colorless oil with the molecular formula of C8H12O5 as elucidated by the HRESIMS m/z 187.0610 [M − H] − (calcd for C8H11O5, 187.0601). The IR absorption bands at 3373, 1739 and 1652 cm -1 suggested the presence of hydroxyl, α,β-unsaturated lactone groups. The 1 H NMR spectrum in MeOH-d4 displayed signals for a singlet olefinic proton at δH 5.86, an oxymethine at δH 3.56 (dd, J = 7.0, 3.2 Hz), a diastereotopic methylene
Compound 2 was also isolated as an optically inactive colorless oil with the molecular formula of C 8 3.66 (dd, J = 11.7, 7.0 Hz), a methoxyl at δ H 3.50 (s) and a methyl at δ H 2.09 (Table 1). The 13 C NMR spectrum (Table 1), with the help of DEPT and HSQC data, showed the presence of two methyls (one oxygenated), an oxymethylene, an olefinic and an oxymethine, a hemiketal carbon (δ C 110.1), and two quaternary carbons (δ C 172.9 and 168.7). The above data indicated 2 was also a γ-hydroxyl butenolide as 1. The main differences were the appearances of an oxygenated methyl, an oxymethylene and an oxymethine, together with the disappearances of a carbonyl and a methyl. The planar structure and the assignments of the NMR data were completed by the HMBC correlations ( Figure 2). Since no optical rotation and no CD absorption exhibited, compound 2 was also an inseparable racemic mixture.
Molecules 2019, 24, x FOR PEER REVIEW 3 of 13 at δH 3.90 (m) and 3.66 (dd, J = 11.7, 7.0 Hz), a methoxyl at δH 3.50 (s) and a methyl at δH 2.09 ( Table  1). The 13 C NMR spectrum (Table 1), with the help of DEPT and HSQC data, showed the presence of two methyls (one oxygenated), an oxymethylene, an olefinic and an oxymethine, a hemiketal carbon (δC 110.1), and two quaternary carbons (δC 172.9 and 168.7). The above data indicated 2 was also a γ-hydroxyl butenolide as 1. The main differences were the appearances of an oxygenated methyl, an oxymethylene and an oxymethine, together with the disappearances of a carbonyl and a methyl. The planar structure and the assignments of the NMR data were completed by the HMBC correlations ( Figure 2). Since no optical rotation and no CD absorption exhibited, compound 2 was also an inseparable racemic mixture.   293.1020) gave the molecular formula of C15H18O6. The 1 H NMR spectrum displayed signals for two meta-coupled aromatic protons at δH 6.38 and 6.20 (d, J = 1.6 Hz, each), one isolated olefinic proton at δH 5.20, a set of nonequivalent methylene protons at δH 2.58 and 2.08, one methine at δH 2.35 (m), one methoxyl at δH 3.94 and two methyl protons at δH 2.19 and 0.86 ( Table 2). The 13 C NMR spectrum displayed fifteen resonance signals ( Table 2). The carbon signals at δC 180.9, 170.7, 106.3, 90.2 and 60.0 showed the representative resonances for a γ-hydroxyl-β-methoxyl butenolide moiety as in dihydropenicillic acid [20], which was also isolated in the current report. Obviously, two aromatic methines and four aromatic quaternary carbons constructed a tetrasubstituted phenyl ring, and the linkages of substituents were completed by the HMBC correlations ( Figure 2). The splitting behaviors of methylene at δH 2.58 (dd, J = 13.6, 11.2 Hz) and 2.08 (disturbed by the signals of solvent), methine at δH 2.35 (m) and methyl at δH 0.86 (br s) completed the structure of 3. The weak carbon signals of C-4, C-6, C-7, and C-14, together with the broad singlet of H-14 (should be doublet) ( Figure S15 and S16), implied that the C-4 anomers   Table 2). The 13 C NMR spectrum displayed fifteen resonance signals ( Table 2). The carbon signals at δ C 180.9, 170.7, 106.3, 90.2 and 60.0 showed the representative resonances for a γ-hydroxyl-β-methoxyl butenolide moiety as in dihydropenicillic acid [20], which was also isolated in the current report. Obviously, two aromatic methines and four aromatic quaternary carbons constructed a tetrasubstituted phenyl ring, and the linkages of substituents were completed by the HMBC correlations ( Figure 2). The splitting behaviors of methylene at δ H 2.58 (dd, J = 13.6, 11.2 Hz) and 2.08 (disturbed by the signals of solvent), methine at δ H 2.35 (m) and methyl at δ H 0.86 (br s) completed the structure of 3. The weak carbon signals of C-4, C-6, C-7, and C-14, together with the broad singlet of H-14 (should be doublet)  Figure S15 and S16), implied that the C-4 anomers mutually transformed in solution. Owing to no optical rotation and no CD absorption displayed, 3 existed as an inseparable racemic mixture. O unit less than that of 3. Its 1 H and 13 C NMR data were similar to those of 3, except for the chemical shifts of C-4, C/H-5, C/H-6, C-7 and C-8 ( Table 2). The above information, especially the chemical shift of C-8 (δ C 153.2), implied that 8-OH and 4-OH in 3 should be dehydrated into an ether linkage in 4. The 2D NMR spectra ( Figure 2) established its planar structure, which was different from aspergispiroketal in the locations of the substituents at benzyl moiety [21], as proved by the HMBC correlations from H-6 to C-7 and C-8, from H-9 to C-8, C-10 and C-11, from H-11 to C-7, C-9, C-10 and C-13, and from H-13 to C-7, C-11 and C-12. In view of no optical activity and ECD absorption, 4 was a racemate and separated into (+)-4 and (-)-4 using high performance liquid chromatography (HPLC) on a chiral column. Their absolute configurations were determined according to the experimental and calculated ECD data ( Figure 3). Based on the optimized structures, the ECD calculation was conducted using time-dependent density functional theory (TD-DFT) at BP86/6-311G (d,p) for four isomers of 4 (Attachment S1). Then the absolute configurations of (+)-4 and (-)-4 were determined to be (4S,5R)-4b and (4R,5S)-4a, respectively.
Molecules 2019, 24, x FOR PEER REVIEW 4 of 13 mutually transformed in solution. Owing to no optical rotation and no CD absorption displayed, 3 existed as an inseparable racemic mixture. Compound 4 was afforded as an optically inactive colorless solid. The HRESIMS m/z 277.1071 [M + H] + gave a molecular formula of C15H16O5, which was one H2O unit less than that of 3. Its 1 H and 13 C NMR data were similar to those of 3, except for the chemical shifts of C-4, C/H-5, C/H-6, C-7 and C-8 ( Table 2). The above information, especially the chemical shift of C-8 (δC 153.2), implied that 8-OH and 4-OH in 3 should be dehydrated into an ether linkage in 4. The 2D NMR spectra ( Figure 2) established its planar structure, which was different from aspergispiroketal in the locations of the substituents at benzyl moiety [21], as proved by the HMBC correlations from H-6 to C-7 and C-8, from H-9 to C-8, C-10 and C-11, from H-11 to C-7, C-9, C-10 and C-13, and from H-13 to C-7, C-11 and C-12. In view of no optical activity and ECD absorption, 4 was a racemate and separated into (+)-4 and (-)-4 using high performance liquid chromatography (HPLC) on a chiral column. Their absolute configurations were determined according to the experimental and calculated ECD data ( Figure 3). Based on the optimized structures, the ECD calculation was conducted using time-dependent density functional theory (TD-DFT) at BP86/6-311G (d,p) for four isomers of 4 (Attachment S1). Then the absolute configurations of (+)-4 and (-)-4 were determined to be (4S,5R)-4b and (4R,5S)-4a, respectively. Experimental ECD of (+)-5 Experimental ECD of (-)-5 Calculated ECD of (4S,5S)-5a Calculated ECD of (4R,5R)-5b  Compound 5, a colorless solid with zero optical rotation, was assigned the molecular formula of C 7 H 10 O 3 on the basis of its positive HRESIMS at m/z 143.0704 [M + H] + (calcd for C 7 H 11 O 3 , 143.0703). The 1 H NMR spectrum showed an olefinic proton at δ H 6.37 (d, J = 2.6 Hz), a oxymethine at δ H 4.37 (dd J = 2.6, 1.6 Hz), a methine at δ H 2.20 (qd, J = 7.5, 1.6 Hz), a methoxyl at δ H 3.75 (s) and a doublet methyl at δ H 1.20 (J = 7.5 Hz) ( Table 1). The 13 C NMR spectrum displayed seven carbon signals including a keto carbonyl, two olefinic and four aliphatic carbons ( Table 1). The planar structure of 5 was confirmed by the HMBC correlations ( Figure 2). In the NOE difference experiment, the signal of H-2 was enhanced when H-6 was irradiated, so H-2 and H-3 were in the opposite directions, which was further confirmed by the small coupling constant (J = 1.6 Hz). Compound 5 was subjected to chiral HPLC and separated into (+)-5 and (−)-5. According to the calculated and experimental ECD data (Figure 3), their absolute configurations were determined to be 2S,3S and 2R,3R for 5a and 5b, respectively. 5b had been reported as a synthetic intermediate without 13 C NMR, ECD and specific rotation data reported [22]. Therefore, 5a was a new compound, while 5b was a new natural product.
Compound 6 was obtained as colorless blocks with the molecular formula of C 8 H 12 O 5 on the basis of its negative HRESIMS. Some articles reported its structure with a set of 1 H and 13 C NMR data [4,23], but the compound obtained in our project showed two sets of 1 H and 13 C NMR data (6a and 6b) ( Table 3) with the ratio of about 1:1 in DMSO-d 6 , about 2:1 in acetone-d 6 (only 1 H NMR spectrum measured, Figure S42), about 1.4:1 in MeOH-d 4 . Additionally, only H-6, H-8, and C-5 exhibited two sets of NMR signals, and signals of C-3 and C-4 were too weak to be observed in MeOH-d 4 ( Figure  S43 and S44). The above phenomena suggested 6 was a mixture of two pairs of racemates, and the proportion of anomers of C-4 changed with solvents ( Figure 4). The structures and the assignments of the 1 H and 13 C NMR data of 6a and 6b in DMSO-d 6 were completed by 2D NMR spectra ( Figure 2). In order to explore the case, compound 6 was reacted with AcCl leading to the production of 6c and 6d with optical inactivity (Figure 4). Their molecular formulae of C 12 H 16 O 7 were obtained on the basis of their HRESIMS. And their planar structures were constructed by the 1 H and 13 C NMR (Table 4) and HMBC spectra ( Figure 4). The single crystal X-ray diffraction using Cu Kα radiation showed 6c to be a centrosymmetric space group P2 1 /C with 4S,5R and 4R,5S configurations ( Figure 5), so (±)-6d should be the 4S,5S and 4R,5R configurations. Then they were subjected to chiral HPLC and isolated into (+)/(−)-6c and (+)/(−)-6d, respectively. The calculated and experimental ECD data ( Figure 6) proved the absolute configurations of (+)-6c and (−)-6c to be the respective 4R,5S and 4S,5R. Considering their ECD absorptions mainly resulted from C-4 chiral center, the same ECD data of (+)-6c and (+)-6d, and (−)-6c and (−)-6d to the mirror images, implied that (+)-6d and (−)-6d had 4R,5R and 4S,5S configurations, respectively. The δ values of C-1, C-2 and C-4 in 6c were slightly larger than those in 6d with C-3 and C-5 to the contrary (Table 4), and the same behaviors were also observed in 6b and 6a (Table 3), so (±)-6c and (±)-6d should derive from 6b and 6a, separately. Consequently, 6b should be a racemate with the configurations of 4S,5R and 4R,5S, 6a be 4S,5S and 4R,5R. and 6b) ( Table 3) with the ratio of about 1:1 in DMSO-d6, about 2:1 in acetone-d6 (only H NMR spectrum measured, Figure S42), about 1.4:1 in MeOH-d4. Additionally, only H-6, H-8, and C-5 exhibited two sets of NMR signals, and signals of C-3 and C-4 were too weak to be observed in MeOH-d4 ( Figure S43 and S44). The above phenomena suggested 6 was a mixture of two pairs of racemates, and the proportion of anomers of C-4 changed with solvents ( Figure 4). The structures and the assignments of the 1 H and 13 C NMR data of 6a and 6b in DMSO-d6 were completed by 2D NMR spectra (Figure 2).  In order to explore the case, compound 6 was reacted with AcCl leading to the production of 6c and 6d with optical inactivity (Figure 4). Their molecular formulae of C12H16O7 were obtained on the basis of their HRESIMS. And their planar structures were constructed by the 1 H and 13 C NMR (Table 4) and HMBC spectra (Figure 4). The single crystal X-ray diffraction using Cu Kα radiation showed 6c to be a centrosymmetric space group P21/C with 4S,5R and 4R,5S configurations ( Figure  5), so (±)-6d should be the 4S,5S and 4R,5R configurations. Then they were subjected to chiral HPLC and isolated into (+)/(−)-6c and (+)/(−)-6d, respectively. The calculated and experimental ECD data ( Figure 6) proved the absolute configurations of (+)-6c and (−)-6c to be the respective 4R,5S and 4S,5R. Considering their ECD absorptions mainly resulted from C-4 chiral center, the same ECD  data [4,23], but the compound obtained in our project showed two sets of 1 H and 13 C NMR data (6a and 6b) ( Table 3) with the ratio of about 1:1 in DMSO-d6, about 2:1 in acetone-d6 (only 1 H NMR spectrum measured, Figure S42), about 1.4:1 in MeOH-d4. Additionally, only H-6, H-8, and C-5 exhibited two sets of NMR signals, and signals of C-3 and C-4 were too weak to be observed in MeOH-d4 ( Figure S43 and S44). The above phenomena suggested 6 was a mixture of two pairs of racemates, and the proportion of anomers of C-4 changed with solvents ( Figure 4). The structures and the assignments of the 1 H and 13 C NMR data of 6a and 6b in DMSO-d6 were completed by 2D NMR spectra ( Figure 2). In order to explore the case, compound 6 was reacted with AcCl leading to the production of 6c and 6d with optical inactivity (Figure 4). Their molecular formulae of C12H16O7 were obtained on the basis of their HRESIMS. And their planar structures were constructed by the 1 H and 13 C NMR (Table 4) and HMBC spectra (Figure 4). The single crystal X-ray diffraction using Cu Kα radiation showed 6c to be a centrosymmetric space group P21/C with 4S,5R and 4R,5S configurations (Figure had 4R,5R and 4S,5S configurations, respectively. The δ values of C-1, C-2 and C-4 in 6c were slightly larger than those in 6d with C-3 and C-5 to the contrary (Table 4), and the same behaviors were also observed in 6b and 6a (Table 3), so (±)-6c and (±)-6d should derive from 6b and 6a, separately. Consequently, 6b should be a racemate with the configurations of 4S,5R and 4R,5S, 6a be 4S,5S and 4R,5R. Compound 7 was isolated as colorless blocks. Its NMR data was almost identical with spersclerotioron G, which was reported as an S configuration compound at C-4 chiral center [4]. However, in our report it was optically inactive and of no Cotton effects in its ECD spectrum, therefore it was a racemic mixture, which was confirmed by a centrosymmetric space group P21/n in the single crystal X-ray diffraction with Cu Kα radiation ( Figure 5). Other known compounds were identified as penicillic acid (8) [20], dihydropenicillic acid (9) [20], orcinol (10) [24], and p-hydroxyl benzaldehyde (11) [25], by comparison of their spectroscopic data with those in the literature.
Meanwhile, the antimicrobial assays of compounds 1-9 were screened against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231). Among them, the known compound 8 showed pronounced antimicrobial activity to the tested organisms, while compounds 2 and 3 displayed weak activity against S. aureus and E. coli (Table 5).

Discussion
A. sclerotiorum JH42 not only produced six new compounds (1-3, 4a, 4b and 5a) and a new natural product (5b), but also penicillic acid (8) with a high yield (59 g in 73 g of the crude extract). Since penicillic acid possessed multiple bioactivities, such as antitumor [19,[26][27][28], antibacterial Compound 7 was isolated as colorless blocks. Its NMR data was almost identical with spersclerotioron G, which was reported as an S configuration compound at C-4 chiral center [4]. However, in our report it was optically inactive and of no Cotton effects in its ECD spectrum, therefore it was a racemic mixture, which was confirmed by a centrosymmetric space group P2 1 /n in the single crystal X-ray diffraction with Cu Kα radiation ( Figure 5). Other known compounds were identified as penicillic acid (8) [20], dihydropenicillic acid (9) [20], orcinol (10) [24], and p-hydroxyl benzaldehyde (11) [25], by comparison of their spectroscopic data with those in the literature. Meanwhile, the antimicrobial assays of compounds 1-9 were screened against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231). Among them, the known compound 8 showed pronounced antimicrobial activity to the tested organisms, while compounds 2 and 3 displayed weak activity against S. aureus and E. coli (Table 5).
Additionally, the γ-hydroxyl butenolides (1-3, 6-7) in this study were all proved to be mixtures of enantiomers. The information implied that γ-hydroxyl butenolides usually exist in a mixture of anomers of C-4, which were inseparable because of their mutual transformation through the γ-keto-acid form. The results have guiding significance for the isolation and structure determination of this kind of compounds.

Fermentation and Extraction
A. sclerotiorum JH42 was cultured on Petri dishes of potato dextrose agar (PDA) at 28 • C for 7 days. A small spoon of spores was transferred into 500-mL conical flasks containing 180 mL culture medium (decoction of 200 g potato, glucose 20 g, maltose 20 g, mannitol 10 g, yeast extract 3 g, KH 2 PO 4 0.5 g, MgSO 4 ·7H 2 O 0.3 g, dissolved in 1 L seawater), and cultured at 28 • C for 9 days on a rotary shaker at 170 rpm. The culture broth (34.5 L) was filtered into filtrate and mycelia. The former was extracted with ethyl acetate, while the latter was extracted with methanol. The methanol solution was concentrated under reduced pressure to yield an aqueous solution, which was then extracted with ethyl acetate. The ethyl acetate extracts were merged and evaporated under reduced pressure to give an extract (73 g).

Biological Assay
The cytotoxic activity against HL60, A549 and HL-7702 cell lines was performed by the MTT method as previously described [33].
The antimicrobial assays against S. aureus, E. coli and C. albicans were carried out by the broth microdilution method [34]. The tested organisms were incubated overnight with shaking (200 rpm) in thermostatic oscillation incubator (37 • C) in Mueller Hinton broth (MHB) and liquid Sabourand medium for the bacteria and the fungus, respectively. The microbial inoculum density was adjusted to 1 × 10 6 cfu/mL with 0.9% saline solution by comparison with a MacFarland standard. The tested substances and positive drugs were dissolved in methanol to an initial concentration of 40 mg/mL. 4 µL of initial compound solution and 196 µL of MHB (liquid Sabourand medium for fungus) were added into the first well and mixed evenly. Then 100 µL of solution from the first hole, along with 2 µL of methanol and 98 µL of MHB (liquid Sabourand medium for fungus), were transferred to the second hole, and then shaken up as mixture uniform. The repetitive operation was performed to the eleventh one, from which 100 µL of solution well was discarded. Then, 100 µL of microbial suspension was added to the solutions in 96-well to achieve a final volume of 200 µL and final sample concentrations from 400 to 0.39 µg/mL. The blank well was also incubated with only medium under the same conditions. All experiments were carried out in triplicate and with chloramphenicol and ketoconazole as the positive controls. Optical density measurement for bacteria and fungus was recorded at 620 nm after incubation at 37 • C for 12 and 24 h, respectively. The minimal inhibitory concentration (MIC) was defined as the concentration at which the growth was inhibited 80% of the tested microorganisms [35].

Conclusions
In summary, three new (1-3) and four known (6-9) γ-hydroxyl butenolides, a pair of new enantiomeric spiro-butenolides (4a and 4b), a pair of enantiomeric cyclopentenones (5a new and 5b new natural), along with orcinol (10) and p-hydroxyl benzaldehyde (11), were isolated from A. sclerotiorum JH42. The acquisition of two pairs of enantiomers [(+)/(−)-6c) and (+)/(−)-6d] by the reaction of 6 with AcCl confirmed that 6 was a mixture of two pairs of enantiomers. In addition, the X-ray diffraction data of 7 revealed it was also a racemic mixture. Compound 1 exhibited moderate cytotoxicity against HL60 and A549 cell lines with IC 50 values of 6.5 and 8.9 µM, respectively. New compounds 2 and 3 showed weak antibacterial activity against S. aureus and E. coli, while 8 displayed pronounced antimicrobial activity against all the tested organisms.