Chemical Constituents of the Deep-Sea-Derived Penicillium solitum

A systematic chemical investigation of the deep-sea-derived fungus Penicillium solitum MCCC 3A00215 resulted in the isolation of one novel polyketide (1), two new alkaloids (2 and 3), and 22 known (4–25) compounds. The structures of the new compounds were established mainly on the basis of exhaustive analysis of 1D and 2D NMR data. Viridicatol (13) displayed moderate anti-tumor activities against PANC-1, Hela, and A549 cells with IC50 values of around 20 μM. Moreover, 13 displayed potent in vitro anti-food allergic activity with an IC50 value of 13 μM, compared to that of 92 μM for the positive control, loratadine, while indole-3-acetic acid methyl ester (9) and penicopeptide A (10) showed moderate effects (IC50 = 50 and 58 μM, respectively).


Introduction
Penicillium solitum is a filamentous fungus associated with the decay of pomaceous fruits during storage [1]. As a matter of fact, it can infect fruit through wounds and cause significant economic losses [2]. Besides pome fruits such as apples and pears, this fungus was also isolated from other foods, including cheeses and processed meats [3,4]. Surprisingly, it can also be found under extremophilic circumstances: in the Berkeley Pit Lake (pH 2.7) [5] and the maritime Antarctic [6].

Results and Discussion
Compound  which constructed a long chain of C-5/C-6/C-7/C-8/C-8a/C-1/C-9/C-10/C-11/C-12/C-13/C-14 and C-1 via C-2 to C-16/C-3/C-4 ( Figure 2). The segment and the methoxyl moiety could be connected on the basis of the HMBC correlations of H-4 to C-8a/C-4a/C-5, H 2 -14 and 15-OMe to C-15 ( Figure 2). Therefore, the planar structure of 1 was established as a methyl ester of acyclic form of ML-236A (5) [13], which was previously prepared in the lab by the saponification of ML-236A in 0.1 N NaOH at 50 • C for 2 h [30]. basis of the HMBC correlations of H-4 to C-8a/C-4a/C-5, H2-14 and 15-OMe to C-15 ( Figure  2). Therefore, the planar structure of 1 was established as a methyl ester of acyclic form of ML-236A (5) [13], which was previously prepared in the lab by the saponification of ML-236A in 0.1 N NaOH at 50 °C for 2 h [30].    basis of the HMBC correlations of H-4 to C-8a/C-4a/C-5, H2-14 and 15-OMe to C-15 ( Figure  2). Therefore, the planar structure of 1 was established as a methyl ester of acyclic form of ML-236A (5) [13], which was previously prepared in the lab by the saponification of ML-236A in 0.1 N NaOH at 50 °C for 2 h [30].      (Figure 4), the same as solitumidine D [10], namely 4, which was simultaneously obtained along with 2 by HPLC using the A4-5 chiral column. Since the specific optical rotation of 2 was +6, opposite to that of 4 (−7) in the same concentration of MeOH (c 0.10), 2 was then deduced to be the enantiomer of 4. Accordingly, 2 was determined as (+)-solitumidine D. fragments could be connected by the HMBC correlations of H3-11/H3-12 to C-2, H-1 to C-2/C-3a/C-7/C-7a, H2-4 to C-3, and H-14 to C-16 to construct the planar structure of 2 (Figure 4), the same as solitumidine D [10], namely 4, which was simultaneously obtained along with 2 by HPLC using the A4-5 chiral column. Since the specific optical rotation of 2 was +6, opposite to that of 4 (−7) in the same concentration of MeOH (c 0.10), 2 was then deduced to be the enantiomer of 4. Accordingly, 2 was determined as (+)-solitumidine D. Furthermore, by extensive analysis of the COSY and HMBC NMR spectra (Fig. 4), 3 was determined as 20-O-methyl solitumidine B. Since the optical rotation value for solitumidine B was −55 in MeOH, while it was 0 for 3 in the same solvent, 3 was supposed to be a racemic mixture. As such, it was subjected to further isolation by HPLC with chiral columns. Yet, 3 seemed to be inseparable as it exhibited only one peak using several different mobile phases in A3-5 and A4-5 chiral columns, the latter of which was utilized to successfully isolate 2 from its enantiomer, 4. On the basis of the above evidence, 3 was then named as (±)-solitumidine E.
Moreover, compounds 1-25 were also tested in vitro for anti-food allergic activity. Indole-3-acetic acid methyl ester (9) and penicopeptide A (10) showed modest activity (IC50 = 50 and 58 μM, respectively), while 13 displayed a potent effect with an IC50 value of 13 μM, compared to that of 92 μM for loratadine, an anti-food allergic medicine in clinic. In fact, viridicatol isolated from another deep-sea-derived fungus, Penicillium griseofulvum MCCC 3A00225, showed a significant anti-food allergic effect in the RBL-2H3 cell model and the ovalbumin-induced food allergy mouse [31] . Therefore, it may represent a novel therapeutic for allergic diseases.

General Experimental Procedures
NMR spectra were recorded on a Bruker 400 MHz spectrometer. The HRESIMS spectra were recorded on a Waters Q-TOF mass spectrometer (Xevo G2). Optical rotations were obtained with an Anton Paar polarimeter (MCP100). ECD spectra were measured on a Chirascan spectrometer. The semi-preparative HPLC was conducted on an Agilent instrument (1260) with different kinds of columns (COSMOSIL 5 C18-MS-II, Nacalai Tesque, Japan; ColumnTek TM Chiral A3-5 and A4-5). Column chromatography was performed on silica gel, Sephadex LH-20, and ODS. Compound 3 presented its molecular formula asC 21 H 28 N 2 O 4 by the positive HRES-IMS at m/z 395.1947 [M + Na] + . The 1 H and 13 C NMR spectra consisted of signals almost the same as those of solitumidine B [10] except for an additional methoxyl unit. This was confirmed by the HMBC correlation of 20-OMe (δ H 3.73, s) to C-20 (δ C 176.0, s). Furthermore, by extensive analysis of the COSY and HMBC NMR spectra (Figure 4), 3 was determined as 20-O-methyl solitumidine B. Since the optical rotation value for solitumidine B was −55 in MeOH, while it was 0 for 3 in the same solvent, 3 was supposed to be a racemic mixture. As such, it was subjected to further isolation by HPLC with chiral columns. Yet, 3 seemed to be inseparable as it exhibited only one peak using several different mobile phases in A3-5 and A4-5 chiral columns, the latter of which was utilized to successfully isolate 2 from its enantiomer, 4. On the basis of the above evidence, 3 was then named as (±)-solitumidine E.
Moreover, compounds 1-25 were also tested in vitro for anti-food allergic activity. Indole-3-acetic acid methyl ester (9) and penicopeptide A (10) showed modest activity (IC 50 = 50 and 58 µM, respectively), while 13 displayed a potent effect with an IC 50 value of 13 µM, compared to that of 92 µM for loratadine, an anti-food allergic medicine in clinic. In fact, viridicatol isolated from another deep-sea-derived fungus, Penicillium griseofulvum MCCC 3A00225, showed a significant anti-food allergic effect in the RBL-2H3 cell model and the ovalbumin-induced food allergy mouse [31]. Therefore, it may represent a novel therapeutic for allergic diseases.

General Experimental Procedures
NMR spectra were recorded on a Bruker 400 MHz spectrometer. The HRESIMS spectra were recorded on a Waters Q-TOF mass spectrometer (Xevo G2). Optical rotations were obtained with an Anton Paar polarimeter (MCP100). ECD spectra were measured on a Chirascan spectrometer. The semi-preparative HPLC was conducted on an Agilent instrument (1260) with different kinds of columns (COSMOSIL 5 C18-MS-II, Nacalai Tesque, Japan; ColumnTek TM Chiral A3-5 and A4-5). Column chromatography was performed on silica gel, Sephadex LH-20, and ODS.

Fungal Identification, Fermentation, and Extract
The fungus Penicillium solitum MCCC 3A00215 was isolated from a sediment sample of the Northwest Atlantic Ocean (−3034 m, W 44.9801 • , N 14.7532 • ). For the large-scale fermentation procedure, see our recently published literature [12]. The crude extract (200 g) was subjected to column chromatography on silica gel using petroleum ether (PE), CH 2 Cl 2 , EtOAc to provide a CH 2 Cl 2 -soluble extract (11 g) and a EtOAc-soluble extract (114.5 g), respectively.

Isolation and Purification
The CH 2 Cl 2 crude extract was separated into six fractions (Fr.A−Fr.F) via medium pressure liquid chromatography (MPLC, 460 mm × 36 mm) with gradient PE-EtOAc

ECD Calculation
Conformational analysis was performed by the Sybyl-X 2.0 using the MMFF94S force field as reported [32]. Predominant conformers were relocated and confirmed at the B3LYP/6-31G(d) level. The theoretical ECD spectra were calculated with the timedependent density functional theory (TD-DFT) in acetonitrile. The ECD spectrum was obtained by averaging each conformer using the Boltzmann distribution theory.

Cell Proliferation Assay
Cytotoxic activities of all isolates were conducted on 17 human tumor cell lines of A431, A549, MB231, MCF-7, PANC1, HepG2, HCT116, H460, H1299, QGY-7701, BGC823, SKGT4, A375, U2OS, HL-60, K562, and KYSE450 by the MTT method [33]. Paclitaxel was used as a positive control, and DMSO was used as a negative control. Different cancer cells were incubated on 96-well cell plates and cultured for 24 h. Thereafter, the cells were treated with different concentrations of tested compounds and controls. After 48 h, MTT (20 µL) was added to incubate for another 4 h. The supernatant was discarded softly, and the deposited formazan formed in the cells was dissolved with DMSO (100 µL). The absorbencies were measured at 490 nm.

Anti-Allergic Bioassay
The in vitro anti-food allergic experiment was performed as previously reported [32]. In brief, rat basophilic leukemia 2H3 (RBL-2H3) cells were incubated with dinitrophenyl (DNP)-immunoglobulin E (IgE) overnight. Then, the IgE-sensitized RBL-2H3 cells were pretreated with tested compounds and stimulated with DNP-bovine serum albumin (BSA). The bioactivity was quantified by measuring the fluorescence intensity of the hydrolyzed substrate in a fluorometer. Loratadine, a commercially available antiallergic medicine, was used as a positive control.