New dibenzodioxocinone and pyran-3,5-dione derivatives from the deep-sea-derived fungus Penicillium canescens SCSIO z053

Abstract A new isopentylated dibenzodioxocinone, canescenin A (1), and a new isopentylated pyran-3,5-dione derivative, canescenin B (2), were isolated from an extract of the deep-sea-derived fungus Penicillium canescens SCSIO z053. Their structures were elucidated by spectroscopic analysis. It was rare to obtain pyran-3,5-dione derivatives from nature. Antibacterial, cytotoxic, and antiviral activities of 1 and 2 were also evaluated. Graphical Abstract


Introduction
Penicillium canescens is known to be a sediment associated fungus which can degrade plant matter through the production of abundant xylanases and glucosidases [1]. Previous natural product related studies on this fungus have isolated compounds with antibiotic and antifungal properties including antibiotic canescin [2], antifungal tetrapeptide D-Phe-L-Val-D-Val-L-Tyr, diketopiperazines, and pseurotin A [3,4]. In this study, the P. canescens SCSIO z053 strain was isolated from a deep-sea sediment collected from Okinawa Trough (27 33.07 0 N and 126 58.36 0 E, $1387 m depth) that was about 0.4 km away from an active hydrothermal vent. Further study on the fermentation extract of the strain led to the obtainment of a new isopentylated dibenzodioxocinone, canescenin A (1), and a new isopentylated pyran-3,5-dione derivative, canescenin B (2) (Figure 1). Antibacterial, cytotoxic, and antiviral activities of 1 and 2 were also preliminarily evaluated. Herein, we describe the isolation and structure elucidation of 1 and 2 and their bioactivities.

Results and discussion
The ethyl acetate extract of P. canescens SCSIO z053 was subjected to various chromatographic techniques, including silica gel, Sephadex LH-20, ODS, and semipreparative reversed phase HPLC to give compounds 1 and 2.
Canescenin A (1) was a white amorphous solid and had a molecular formula of C 23 6), and two carboxyl groups (d C 167.0, 169.6). These data indicated that 1 contained a 1,2,3,5-tetrasubstituted benzene ring (A-ring) and a 1,2,3,4-tetrasubstituented benzene ring (B-ring), which was proved by the HMBC and 1 H-1 H COSY spectra ( Figure 2). In addition, the HMBC spectrum showed correlations of H-15 with C-2/C-3/C-16/C-17/C-20, H-16 with C-3/C-17/C-18/C-19, H-18 and H-19 with C-17, and H-21 with C-20, and the COSY spectrum showed correlation of H-15 with H-16, which suggested an acetyl group attached on C-15 and an acetylated isopentyl group attached on C-3 of the B-ring. Furthermore, combining the molecular formula of C 23 H 26 O 8 , the HMBC spectrum showing correlations of H-5 with C-7 and H-8 with C-7/C-9/C-10/C-14 suggested that A-and B-rings were connected by the two fragments of -C(1)-O-C(10)-and -C(6)-CO-CH 2 -C(9)-to form an eight-member heterocyclic unit. So, the planar structure of 1 was determined as shown, which was similar to the structures of purpactins A-C [5] and penicillide [6]. It was reported that this kind of dibenzodioxocinone was a new class of inhibition of cholesterol ester transfer protein (CETP) [7]. We have tried to determine the absolute configuration of C-15 in 1 by using calculated ECD spectra. However, the Cotton effect of the experimental ECD spectrum of 1 was very weak (see Supporting Information Figure S9). As a solution, we performed an esterification reaction to obtain a monoacylation product of 1 (1a) at the C-11 of benzene ring (A-ring) and tried to get a monocrystal of the product for determining the absolute configuration of 1. Unfortunately, we failed to get a monocrystal of 1a.
Compounds 1 and 2 were tested for their antibacterial and antibiofilm activities toward Bascillus amyloliquefaciens and Pseudomonas aeruginosa. Both of the compounds showed weak antibacterial and antibiofilm activities toward B. amyloliquefaciens and P. aeruginosa at 100 lM. Cytotoxicity toward Vero cell line and antiviral activity toward HSV-1 of 1 and 2 were evaluated using MTT methods and a plaque reduction assay, respectively. The results showed that 1 and 2 had no cytotoxicity toward Vero cell under the concentrations of 25.0 lM and had no antiviral activity toward HSV-1 under their noncytotoxic concentrations.

Fungal materials
Penicillium canescens SCSIO z053 registered under Genbank Accession No. JN585930 was isolated from a deep-sea sediment collected from Okinawa Trough (27 33.07 0 N and 126 58.36 0 E, $1387 m depth) that was about 0.4 km away from an active hydrothermal vent in Iheya Ridge. The strain was identified according to ITS rDNA sequence and deposited in the RNAM Center, South China Sea Institute of Oceanology, Chinese Academy of Science.

Fermentation and extraction
Dormant stage P. canescens SCSIO z053 was activated in a potato-dextrose agar (PDA) solid culture. It was then inoculated into 500 ml flasks containing 150 ml liquid growth media consisting of 20% potato broth (200 g/1000 ml), 3% sea salt, 2% glucose, pH 6.5-7.0. This seed culture was incubated in a rotary shaker for 3 days at 28 C. The prepared cell suspension was used to inoculate the large scale culture. 10 ml from the cell suspension was transferred into each 1000 ml flask containing 300 ml liquid medium similar to the seed liquid growth medium containing the similar percentages of nutrients and similar pH level. 25 L of the large scale P. canescens culture was fermented for 25 days at 28 C under stable conditions. At the end of the incubation period, the 25 l fermentation was filtered to separate the mycelia and the broth supernatant. The following extractions were done separately for the broth and the mycelia. The broth was extracted directly using EtOAc. The broth was mixed with EtOAc/H 2 O (v/v 1:1) in a separatory funnel and placed under static conditions to let the organic compounds to seep into the EtOAc solution. Each part of the broth was extracted at least three times with the EtOAc solution to ensure better separation. The EtOAc extraction was then concentrated to get the crude extract. The mycelia were initially extracted with acetone. The acetone solution with the mycelium extraction was then extracted with EtOAc following the same procedure as the broth. Both EtOAc extractions were then concentrated on a rotary evaporator to yield the total crude extract (24 g).

Esterification reaction of 1
Compound 1 (6.0 mg) was dissolved in 1 ml of pyridine-d 5 , it was added 6.0 mg of 4bromobenzoyl chloride, 4-dimethylaminopyridine (1.0 mg), 2-3 drops of trimethylamine, and the reactants were mixed in an airtight round bottom flask. The reaction was carried out for 24 h at room temperature in an air tight container with continuous stirring. At the end of the reaction period, the reaction was stopped by introducing a drop of water to the reaction mixture. The mixture was then freeze dried to remove water and the solvent pyridine and dissolved in methanol. Purification of the reaction mixture was carried out by a YMC-Pack ODS-A column (250 Â 10 mm, 5 lm) with a constant concentration of 80% acetonitrile solution in water over 45 min. The desired monoacylation product was prepared, at the retention time of 30 min to obtain compound 1a. 1a: 13

Computational methods
Conformational searches were performed by employing a systematic procedure implemented in Spartan'14 software package using Molecular Merck force field (MMFF) (Wavefunction, Inc., Irvine, CA, 2013). DFT/TDDFT calculations were conducted using the Guassian09 program (Gaussian, Inc., Pittsburgh PA, 2011). The MMFF conformations were reoptimized to afford low-energy conformers within a 10 kcal/ mol energy window, using the density functional theory (DFT) calculation at the B3LYP/6-31G (d) level using the Guassians09 program. Vibrational frequency calculations were run at the same level to estimate their relative thermal free energies (DG) at 298.15K. A series of single-point energy calculations for the conformers above were performed at the M06-2X/def2-TZVP level, supposing methanol as the solvent with the polarizable continuum model (PCM). The DFT optimized conformers with the Boltzmann distribution over 1% was then subjected to TDDFT calculations using the functional PBE1PBE and basis set 6-311G (d). ECD spectra were generated using the program SpecDis by applying a Gaussian band shape with an exponential halfwidth of 0.2-0.35 eV from dipole-length dipolar and rotational strength. The spectra of the conformers were combined using Boltzmann weighing, with the lowest-energy conformation.

Antibacterial and antibiofilm assay
Antibacterial assay experiments were carried out in compounds 1 and 2 toward two bacterial strains B. amyloliquefaciens and P. aeruginosa [10].

Plaque reduction assays
The cytotoxic activity was evaluated using Vero cell lines by an MTT method. The anti-HSV-1 activity was determined by a plaque assay using monolayer cultures of Vero cells in 24-well culture plates [11].

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The authors are grateful for the financial support provided by the Natural Science Foundation of China (81673326), Strategic Leading Special Science and Technology Program of Chinese