Sporulosol, a New Ketal from the Fungus Paraconiothyrium sporulosum

Sporulosol (1), a new ketal, together with four known compounds, has been isolated from the liquid fermentation cultures of a wetland-soil-derived fungus, Paraconiothyrium sporulosum. Its structure was elucidated primarily by NMR experiments, and was further confirmed by X-ray crystallography. Sporulosol was obtained as a racemic mixture and the resolved two enantiomers racemized immediately after chiral separation. Sporulosol appears to be the first ketal derived from a 6H-benzo[c]chromen-6-one and a benzofuranone unit. The compound showed modest cytotoxicity toward the human tumor cell line T24, with an IC50 value of 18.2 µM.


Introduction
Fungi are one of the most important sources of new drugs or lead compounds; many important medicines have been discovered directly from fungi or developed based on their secondary metabolites [1], such as the antibiotic penicillin, anti-cholesteremic agent lovastatin, immuno-suppressant cyclosporin A, and the anti-multiple sclerosis fingolimod. Fungal species isolated from the wetland environments have attracted increasing attention due to their ability to produce a variety of bioactive secondary metabolites. Examples include stachybisbins A and B [2], flaviphenalenones A-C, aspulvinones P and Q, methybutyrolactone III [3,4], 5-hydroxymethylasterric acid, 3,5-dichlorosulochrin [4], and isocoumarin glycosides [5]. Based on this consideration, we initiated chemical studies of the wetland-soil-derived fungal species in the genus of Paraconiothyrium, which was reclassified as a separate genus as Paraconiothyrium by Verkley in 2004 [6]. Prior to our chemical studies, a variety of bioactive secondary metabolites were reported from this genus [7][8][9][10][11][12][13][14][15][16][17]. Our chemical investigations of P. brasiliense, and P. hawaiiense grown in solid-substrate fermentation cultures have also resulted in the isolation of structurally diverse and biologically active natural products including brasilamides A-N and hawaiinolides A-G [18][19][20][21][22]. During an ongoing search for new bioactive secondary metabolites from the species of this genus, a strain of Paraconiothyrium sporulosum, which was collected at Poyang Lake, Jiangxi Province, People's Republic of China, was subjected to our chemical investigation. A new ketal, named sporulosol (1), was isolated from the liquid fermentation cultures of the fungus, together with four known compounds (2-5; Figure 1). Details of the isolation, structure elucidation, and cytotoxicity of these compounds are reported herein.

Results and Discussion
Sporulosol (1) was assigned a molecular formula of C 26 H 22 O 8 (16 degrees of unsaturation) on the basis of high-resolution electrospray ionization mass spectrometry (HRESIMS). Analysis of its nuclear magnetic resonance (NMR) data (Table 1) revealed the presence of an exchangeable proton (δ H 11.89), five methyl groups including two O-methyls, one ketal carbon (δ C 105.4), 18 aromatic/olefinic carbons with six protonated, one carboxylic carbon (δ C 164.4), and one α,β-unsaturated ketone carbon (δ C 195.9). These data accounted for all the NMR resonances and suggested that 1 was a pentacyclic metabolite. The 1 H and 13 C NMR data of 1 (Table 1) revealed structural features that were closely related to the known compounds graphislactone A (3) [23] and 2-hydroxy-2,4-dimethyl-3(2H)-benzofuranone [24], but some chemical shifts for 1 were significantly different from those reported for the known compounds, warranting detailed 2D NMR analysis. Interpretation of the NMR data revealed a tetrasubstituted aryl ring (A), with a hydroxy and an O-methyl group attached to C-7 and C-9, respectively, which was confirmed by the HMBC correlations from H-8 to C-6a, C-7, and C-10a; H-10 to C-6a, C-9, and C-10a; OH-7 to C-7 and C-8; and from H 3 -11 to C-9. Correlations from H-2 to C-1, C-3, C-4, C-4a, and C-10b; H 3 -13 to C-1 and C-2; and from H 3 -12 to C-3 established the pentasubstituted aryl ring (C). In turn, the HMBC cross peaks from H-4 to C-5 , C-6 , and C-7 ; H-5 to C-4 and C-6 a; H-6 to C-2 a and C-5 ; and from H 3 -7 to C-2 a, C-3 , and C-4 enabled junction of the trisubstituted aryl ring (E) with a methyl group attached to C-3 . Additional correlations from H 3 -8 to C-1 and C-2 , plus the 13 C NMR chemical shifts for C-2 a (δ C 117.4) and C-6 a (δ C 170.0), indicated that these three carbons form an α,β-unsaturated ketone with C-1 connected to C-6 a via the C-1 /C-6 a ether linkage. Considering the 1 H NMR chemical shift of OH-7 (δ H 11.89) and the unsaturation requirement of 1, the carboxylic carbon (δ C 164.4) was connected to C-6, and acylated the C-4a oxygen to form ring B, while C-4 and C-1 were attached to the only remaining oxygen atom to form an ester linkage. Based on these data, the planar structure of 1 was established.
The proposed structure was confirmed by single-crystal X-ray diffraction analysis using Cu Kα radiation, and a perspective ORTEP plot is shown in Figure 2. Compound 1 was found to crystallize as a mixture of 1 S and 1 R enantiomers, which explained the low optical rotation value of +1.0 ([α] 25 D ; c 0.10, MeOH) measured for 1. Subsequently, the racemic mixture was resolved into two enantiomers (1a and 1b; 1:1) and decomposition product graphislactone A (3) using chiral stationary phase (4.6 × 250 mm; 4% 2-propanol in hexanes for 60 min; 0.8 mL/min) ( Figure S7). However, the other decomposition product 2-hydroxy-2,4-dimethyl-3(2H)-benzofuranone was not detected during resolution. The absolute configuration of C-1 was assigned by the application of Snatzke's chirality rule for cyclopentenones [25], through which the negative or positive Cotton effect for n → π * transition in the 330-365 nm region of the CD spectrum was used to assign the 1 S (1a) and 1 R (1b) absolute configurations by HPLC-CD analysis ( Figure 3). Analysis of each collected peak for 1 S (1a) and 1 R (1b) revealed the presence of both enantiomers, suggesting the occurrence of spontaneous equilibration. Similarly, the co-isolated known compound, enalin A (5) [26], was also separated into two enantiomers 5a and 5b in a ratio of 1:1, and the resolved enantiomers again racemized immediately after chiral separation ( Figure S8). However, subsequent HPLC-CD analysis of 5 was unsuccessful, possibly due to the poor HPLC and CD behavior of the compound ( Figure S9). To our knowledge, fungal natural products including isopestacin, pestacin, pestalachloride A, fimetarone A, and arugosins K−M, have been reported as racemic mixtures of the S and R enantiomers [27][28][29][30][31]. The proposed structure was confirmed by single-crystal X-ray diffraction analysis using Cu Kα radiation, and a perspective ORTEP plot is shown in Figure 2. Compound 1 was found to crystallize as a mixture of 1′S and 1′R enantiomers, which explained the low optical rotation value of +1.0 ([α] 25 D ; c 0.10, MeOH) measured for 1. Subsequently, the racemic mixture was resolved into two enantiomers (1a and 1b; 1:1) and decomposition product graphislactone A (3) using chiral stationary phase (4.6 × 250 mm; 4% 2-propanol in hexanes for 60 min; 0.8 mL/min) ( Figure S7). However, the other decomposition product 2-hydroxy-2,4-dimethyl-3(2H)-benzofuranone was not detected during resolution. The absolute configuration of C-1′ was assigned by the application of Snatzke's chirality rule for cyclopentenones [25], through which the negative or positive Cotton effect for n → π * transition in the 330-365 nm region of the CD spectrum was used to assign the 1′S (1a) and 1′R (1b) absolute configurations by HPLC-CD analysis ( Figure 3). Analysis of each collected peak for 1′S (1a) and 1′R (1b) revealed the presence of both enantiomers, suggesting the occurrence of spontaneous equilibration. Similarly, the co-isolated known compound, enalin A (5) [26], was also separated into two enantiomers 5a and 5b in a ratio of 1:1, and the resolved enantiomers again racemized immediately after chiral separation ( Figure S8). However, subsequent HPLC-CD analysis of 5 was unsuccessful, possibly due to the poor HPLC and CD behavior of the compound ( Figure  S9). To our knowledge, fungal natural products including isopestacin, pestacin, pestalachloride A, fimetarone A, and arugosins K−M, have been reported as racemic mixtures of the S and R enantiomers [27][28][29][30][31].   The molecular formula of 2 was determined to be C 15 H 12 O 6 by HRESIMS. Analysis of the 1 H and 13 C NMR data of 2 (Table 1) revealed structural features similar to the known compound graphislactone A (3) [23], except that the 9-OMe signal disappeared, which was verified by interpretation of the 2D NMR data. Compound 2 was the first naturally occurring 4,7,9-trihydroxy-3methoxy-1-methyl-6H-benzo[c]chromen-6-one [32], and its NMR data have yet to be reported. Compounds 3-5 were readily identified as graphislactone A (3) [23], 7,9-dihydroxy-3-methoxy-1methyl-6H-benzo[c]chromen-6-one (4) [33], and enalin A (5) [26] by comparison of their NMR and MS data with those reported.
To verify that 1 was authentic natural product, a portion of the EtOAc extract freshly prepared from the freeze-dried fermented liquid substrate was subjected to HPLC-MS analysis using HPLC-grade H 2 O and CH 3 CN as solvents. Compound 1 was identified on the HPLC-MS chromatogram of the crude extract by comparison of its retention time and ESIMS data with an authentic sample ( Figure S10), indicating that 1 is indeed a naturally occurring metabolite.
Compounds 1-4 were tested for cytotoxicity against a panel of five human tumor cell lines: HeLa, T24, A549, HCT116, and SH-SY5Y (Table 2)  Although sporulosol (1) is a ketal derived from two known metabolites, it is the first example of such a ketal originating from the 6H-benzo[c]chromen-6-one and benzofuranone moieties, representing a new type of chemical structure. The presence of such a ketal moiety generated two enantiomers racemized constantly even right after chiral resolution. In addition, the plausible biosynthetic precursor was identified as a racemic mixture, too. The proposed precursors and the reaction cascades leading to the generation of 1 are illustrated in Scheme 1. The molecular formula of 2 was determined to be C15H12O6 by HRESIMS. Analysis of the 1 H and 13 C NMR data of 2 (Table 1) revealed structural features similar to the known compound graphislactone A (3) [23], except that the 9-OMe signal disappeared, which was verified by interpretation of the 2D NMR data. Compound 2 was the first naturally occurring 4,7,9-trihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one [32], and its NMR data have yet to be reported. Compounds 3-5 were readily identified as graphislactone A (3) [23], 7,9-dihydroxy-3-methoxy-1-methyl-6H-benzo[c]chromen-6-one (4) [33], and enalin A (5) [26] by comparison of their NMR and MS data with those reported.
To verify that 1 was authentic natural product, a portion of the EtOAc extract freshly prepared from the freeze-dried fermented liquid substrate was subjected to HPLC-MS analysis using HPLC-grade H2O and CH3CN as solvents. Compound 1 was identified on the HPLC-MS chromatogram of the crude extract by comparison of its retention time and ESIMS data with an authentic sample ( Figure S10), indicating that 1 is indeed a naturally occurring metabolite.
Compounds 1-4 were tested for cytotoxicity against a panel of five human tumor cell lines: HeLa, T24, A549, HCT116, and SH-SY5Y (Table 2)  Although sporulosol (1) is a ketal derived from two known metabolites, it is the first example of such a ketal originating from the 6H-benzo[c]chromen-6-one and benzofuranone moieties, representing a new type of chemical structure. The presence of such a ketal moiety generated two enantiomers racemized constantly even right after chiral resolution. In addition, the plausible biosynthetic precursor was identified as a racemic mixture, too. The proposed precursors and the reaction cascades leading to the generation of 1 are illustrated in Scheme 1.

General Experimental Procedures
An Analytical Automatic Polarimeter (Rodolph Research) was used to record optical rotations of the isolated compounds, and a Shimadzu Biospec-1601 spectrophotometer was used to measure the ultraviolet (UV) spectra. A Nicolet Magna-IR 750 spectrophotometer was used to record the infrared (IR) spectra. Nuclear magnetic resonance (NMR) spectra were recorded on Inova-400 and Inova-600 spectrometers using TMS as an internal standard. The HMBC and HMQC experiments were optimized for 8.0 and 145.0 Hz, respectively. Electrospray ionization mass spectrometry (ESIMS) and high-resolution mass spectrometry (HRMS) data were measured on an Agilent accurate mass quadrupole time-of-flight LC/MS G6550 instrument. High-performance liquid chromatography-circular dichroism (HPLC-CD) chromatograms were recorded on a JASCO LC 2000-CD 2095 instrument. All HPLC analysis and separation were performed using an Agilent 1260 instrument (Agilent, Santa Clara, CA, USA) equipped with a variable-wavelength UV detector.

Fungal Material
The strain P. sporulosum Verkley was isolated from the soil samples that were collected at Poyang Lake, Jiangxi Province, P. R. China, in December 2010. The fungus was identified by morphological observation and sequence (Genbank Accession No. JX077030) analyses of the ITS region of the rDNA. The identified P. sporulosum strain was cultured on Potato Dextrose Agar (PDA) at room temperature for 10 days, and the resulting agar plugs were cut into small pieces (0.5 × 0.5 × 0.5 cm 3 ) under aseptic conditions. Fifteen pieces were inoculated into three 250 mL Erlenmeyer flasks, each containing 50 mL medium (0.4% glucose, 1% malt extract, and 0.4% yeast extract; pH 6.5), which were then incubated at room temperature on an orbital shaker at 170 rpm for 5 days to prepare the seed culture. The fermentation was carried out in 24 Fernbach flasks of 500 mL, each containing 5.0 mL seed culture and 200 mL synthetic dropout medium (2% malt extract, 6% dextrin, 0.7% peptone form fish, 0.7% cottonseed flour, 0.25% MgSO 4 ·7H 2 O, 0.25% CaCO 3 , 0.1% FeSO 4 ·7H 2 O, and 0.001% ZnSO 4 ·7H 2 O), and incubated at 25 • C on a rotary shaker at 170 rpm for 30 days.

MTT Assay
The cytotoxicity of compounds 1-4 was evaluated with the MTT assay [37]. The cell lines at a density of (2-5) × 10 3 cells/well were seeded in 96-well plates and allowed to adhere for 24 h. Subsequently, compounds 1-4 and cisplatin were added at appropriate concentrations and incubated with cells at 37 • C for 48 h in a 5% CO 2 -containing incubator. Finally, 20 µL of MTS (Promega) was added to each well in the dark to assess the proliferation after 90 min incubation at 37 • C. The optical density was recorded on a microplate reader at 490 nm. All tests were run in triplicate.
Supplementary Materials: The supplementary materials are available online. NMR spectra of Compounds 1 and 2 (Figures S1-S6), Figure S7: HPLC chromatogram of sporulosol (1) using a CHIRALPAK AD-H column; Figure S8: HPLC chromatogram of enalin A (5) using a CHIRALPAK AD-H column title; Figure S9: HPLC-CD chromatogram of enalin A (5) using a CHIRALPAK AD-H column title; Figure S10: HPLC-MS analysis of the crude extract.
Author Contributions: Y.C. and F.R. conceived and designed the experiments; C.Z., P.F., Y.Z. performed the experiments and analyzed the data; X.L. contributed materials; C.Z. and F.R. wrote the paper.