Azaphilones from the Marine Sponge-Derived Fungus Penicillium sclerotiorum OUCMDZ-3839

Four new azaphilones, sclerotiorins A–D (1–4), as well as the dimeric sclerotiorin E (5) of which we first determined its absolute configuration, and 12 known analogues (5–16) were isolated from the fermentation broth of Penicillium sclerotiorum OUCMDZ-3839 associated with a marine sponge Paratetilla sp.. The new structures, including absolute configurations, were elucidated by spectroscopic analyses, optical rotation, ECD spectra, X-ray single-crystal diffraction, and chemical transformations. Compounds 11 and 14 displayed significant inhibitory activity against α-glycosidase, with IC50 values of 17.3 and 166.1 μM, respectively. In addition, compounds 5, 7, 10, 12–14, and 16 showed moderate bioactivity against H1N1 virus.


Results and Discussion
Sclerotiorin A (1) was obtained as a yellow amorphous powder. The molecular formula of 1 was determined to be C 23 H 29 O 4 Cl by the HRESIMS (high resolution electrospray ionization mass spectroscopy) peak at m/z 405.1834 [M + H] + ( Figure S1), with the 1:3 chlorine isotope peaks. 1 H (Table 1, Figure S2), 13 C (Table 2, Figure S3) combined with DEPT (distortionless enhancement by polarization transfer, Figure S4) and HSQC (heteronuclear single-quantum correlation, Figure S5) NMR data of 1 revealed the presence of 5 singlet methyls, 1 methoxy, two methylenes, 2 sp 3 methines, 2 heteroatom-bonded sp 3 non-protonated carbons, 10 olefinic/aromatic carbons, and 1 carbonyl. The HMBC (heteronuclear multiple bond correlation, Figure 2 and Figure S7) from H-1 to C-3 and C-4a, H-4 to C-3, C-5, and C-8a, H-8 to C-1, C-4a, C-6, and C-7 established the core skeleton of azaphilones. 8 Moreover, the COSY (correlation spectroscopy) cross peaks ( Figure 2 and Figure S6) from H-9 to H-10 and from H-13 to H-12, H-14, H-16, then from H-14 to H-15, along with the HMBC correlations from H-9 to C-11, H-10 to C-17 and C-12, H-12 to C-10 and C-17 demonstrated the presence of the common side chain of azaphilones [8]. The linkage of the unsaturated side chain to C-3 was demonstrated by the HMBC correlations from H-9 to C-3 and C-4 along with that from H-10 to C-3. Additionally, the other HMBC correlations from H-20a to C-7, H-20b to C-19, C-8a ,and C-8, H-β-OCH 3 to C-19, and H-21 to C-20 revealed a furan nucleus. The COSY correlations from H-8 to H-20, as well as the HMBC correlations from H-20 to C-7 and C-8, confirmed the connection mode of the furan nucleus to the fused pyrone-quinone core skeleton. Thus, the constitution of sclerotiorin A (1) was determined.
Sclerotiorin B (2) was obtained as a yellow amorphous powder. HRESIMS gave the peak of m/z 405.1836 [M + H] + ( Figure S10) and the chlorine isotope peaks; consequently, the molecular formula was determined to be C 23 H 29 O 4 Cl, the same as that of 1. The NMR data of 2 ( Table 1; Table 2, Figures S11-S14) were similar to those of 1, except that C-8, C-19, and C-20 were shielded and shifted from δ C 43.4, 106.0, and 45.8 of 1 to δ C 42.7, 105.4, and 44.2 of 2, respectively. The chemical shift changes may be triggered by the difference of the stereochemistry on C-19. Accordingly, compound 2 was identified as a 19-epimer of compound 1, which was further confirmed by the 2D NMR data (Figure 2, Figures S15 and S16).    [28], the most obvious differences were that compound 4 had one more methyl group (δ C/H-25 52.2/3.70), and the carbonyl carbon C-24 (δ C 172.6) was deshielded. Consequently, compound 4 was supposed to be the methyl ester derivative of 16. A further analysis of 1D and 2D NMR confirmed the chemical composition of compound 4 ( Figure 2, Figure S31 and Figure S32).
Taking into account the structural similarity of 14 with 4, 5, 7, 15, and 16, the absolute configurations of these compounds could be resolved by chemical correlation if a single crystal of 14 could be obtained. Fortunately, a single crystal of compound 14 was obtained, and the X-ray diffraction ( Figure 4) clearly determined the absolute configuration of 14 as 7R and 13S from the absolute structure parameter of 0.01 (2). Thus, a series of reactions were carried out using (+)-sclerotiorin (7) as a raw material (Scheme 1) [37]. Compounds 14-16 were directly produced after the reaction of 7 with ammonium acetate, aminoethanol, and 3-aminobutyric acid, while compounds 4 and 5 resulted from the reactions of 16 and 14 with iodomethane and 1,4-diiodobutane ( Figure S39), respectively. The synthetic compounds 4, 5, and 14-16 were identified as the natural ones by the same retention times in their co-HPLC profiles, as shown in Figure S37. In addition, compounds 4, 5, and 14-16 displayed similar ECD ( Figure S38) and the same sign of specific rotation. Therefore, compounds 4, 5, and 14-16 had the same (7R,13S) configurations.    Figure S33). Although the constitution of 5 [19] was identified to be the same as that reported by NMR (Table S1 and S2, Figures S34-S37), the absolute configuration has not been resolved yet. Expectedly, compound 5 was the dimer of 14 linked by a 1,4-butylidene bridge. As shown in Scheme 1, compound 5 could be semi-synthesized from 14. Thus, the absolute configuration of compound 5 that we named sclerotiorin E was determined to be (7R, 7 R, 13S, 13 S) for the first time.

Fungal Material and Fermentation
The fungus OUCMDZ-3839 was isolated from Paratetilla sp. sponges collected from Xisha Island in November 2011. The sponges were firstly clipped, then ground and suspended in sterile distilled water. The suspension was spread on a seawater starch (SWS) agar plate (10 g starch, 1 g protein powder, 20 g agar per liter of sea water). After culturing at 28 • C for several days, the single colony was purified on a potato dextrose agar plate (PDA, 200 g potato, 20 g glucose, 20 g agar per liter of tap water) and maintained at −80 • C. After culture on the PDA plate at 28 • C for 4 days, the mycelium was inoculated into 200 × 1000 mL Erlenmeyer flasks, each containing 300 mL of liquid medium (20 g sorbitol, 20 g maltose, 10 g monosodium glutamate, 0.5 g KH 2 PO 4 , 0.3 g MgSO 4 ·7H 2 O, 0.5 g tryptophan, 3 g yeast extract per liter of sea water pH 6.5). The medium was incubated at 28 • C under static conditions for 41 days.

Conversion from 7 to 14
To a solution of compound 7 (15 mg) in 2 ml of THF, 300 mg of NH 4 OAc and 300 µL of MeOH were added. The mixture was stirred at 25 • C for 17 h. The reaction mixture was diluted with 5 mL of water and extracted three times with equal volumes of EtOAc. The EtOAc layers were combined, washed with water, dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to yield a product as an orange oil. The orange oil was purified by semipreparative HPLC on ODS (70% acetonitrile/H 2 O, v/v) to afford compound 14 (6.0 mg, t R 5.6 min, 40% yield) that was identified by the same retention time in the co-HPLC ( Figure S37) and the same sign of specific rotation as natural 14.

Conversion from 7 to 15
Amino ethanol (20 µL) was added to 1 mL of CH 2 Cl 2 solution of compound 7 (6 mg). The reaction solution was stirred at 25 • C for 24 h under a nitrogen atmosphere. After CH 2 Cl 2 was dried in vacuo, the mixture was purified by semipreparative HPLC on an ODS column (85% acetonitrile/H 2 O, v/v) to afford compound 15 (6.1 mg, t R 5.6 min, 92% yield), which was identified by the same retention time in the co-HPLC ( Figure S37) and the same sign of specific rotation as natural 15.

Conversion from 7 to 16
Amino butyric acid (21.5 mg) was added to 2 mL of DMF solution of compound 7 (8 mg). The solution was stirred at 25 • C for 5 h under a nitrogen atmosphere, and then 10 mL of H 2 O was added. The reaction mixture was extracted three times with equal volumes of EtOAc. The EtOAc layers were combined, dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to yield an orange oil. The orange oil was purified by semipreparative HPLC on an ODS column (85% acetonitrile/H 2 O, v/v) to afford compound 16 (9.0 mg, t R 7.9 min, 93% yield), which was identified by the same retention time in the co-HPLC ( Figure S37) and the same sign of specific rotation as natural 16.

Conversion from 14 to 5
1,4-Diiodobutane (6.96 µL) was added to the acetone solution (3 mL) of compound 14 (20 mg) and K 2 CO 3 (21.2 mg). The reaction solution was heated to 50 • C and stirred for 7 days under a nitrogen atmosphere. The reaction solution was concentrated in vacuo and dissolved in 10 mL of H 2 O. The obtained solution was extracted three times with equal volumes of EtOAc. The EtOAc layers were combined, dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to give a gum that was purified by semipreparative HPLC on an ODS column (85% acetonitrile/H 2 O, v/v) to afford compound 5 (1.41 mg, t R 7.9 min, 3.3% yield), which was identified by the same retention time in the co-HPLC ( Figure S37) and the same sign of specific rotation as natural 5.

Conversion from 16 to 4
A volume of 10 µL of CH 3 I was added to the acetone solution of compound 16 (1.33 mg) and K 2 CO 3 (5.50 mg). The solution was stirred at 28 • C for 24 h under a nitrogen atmosphere, and then 10 mL of H 2 O was added. The mixture was extracted three times with equal volumes of CH 2 Cl 2 . After CH 2 Cl 2 was dried in vacuo, the CH 2 Cl 2 extracts were applied to semipreparative HPLC on an ODS column (80% acetonitrile/H 2 O, v/v) to afford compound 4 (1.28 mg, t R 5.40 min, 94% yield), which was identified by the same retention time in the co-HPLC ( Figure S37) and the same sign of specific rotation as natural 4.

Anti-influenza A (H1N1) Virus Bioassay
The antiviral activity against H1N1 was examined by the CPE+MTT assay [38,39]. MDCK cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum in 5% CO 2 at 37 • C. When the cells reached 70-80% confluency, they trypsinized into individual cells, and the cell suspension concentration was adjusted to 1 × 10 5 cells/mL. The cells were seeded in a 96-well plate in 100 µL per well and cultured at 37 • C, 5% CO 2, for 18 h. After eliminating the culture medium, the influenza virus (A/Puerto Rico/8/34 (H1N1), PR/8), diluted to 100 TCID 50 , was added (100 µL per well) to the 96-well plate; an equal amount of virus-free dilution was used as a negative control. The 96-well plate was incubated for 1 h at 37 • C, 5% CO 2 . The samples to be tested and a positive-control drug were diluted in PBS buffer, and 20 µL of these solutions was added into the wells. PBS buffer was used as a negative control. After incubation for 48 h at 37 • C, the cells were fixed with 100 µL of 4% formaldehyde for 20 min at room temperature, then the formaldehyde was poured out, and 50 µL of 0.1% crystal violet stain was added, staining for 30 min at 37 • C. After the plates were washed and dried, the absorbance (OD) of each well was measured at 570 nm with a microplate reader (Bio-Rad, USA). The IC 50 , as the compound concentration required to inhibit influenza virus yield at 48 h post-infection by 50%, was calculated. The IC 50 value of the positive control, Ribavirin, was 179.8 µM.

Anti-α-glycosidase Bioassay
The PNPG method [40] used to evaluate the inhibitory activity against α-glycosidase was described in our previous work [41].

Conflicts of Interest:
The authors declare no conflict of interest.