Cladodionen, a Cytotoxic Hybrid Polyketide from the Marine-Derived Cladosporium sp. OUCMDZ-1635

A new hybrid polyketide, cladodionen (1), together with a new abscisic acid analogue, cladosacid (2), were isolated from the marine-derived fungus, Cladosporium sp. OUCMDZ-1635. Their structures, including the absolute configurations, were fully elucidated on the basis of spectroscopic analysis, ECD spectra, quantum chemical calculations, and chemical methods. Cladodionen (1) showed cytotoxic activities against MCF-7, HeLa, HCT-116, and HL-60 human cancer cell lines with IC50 values of 18.7, 19.1, 17.9, and 9.1 µM.


Introduction
The past decades have witnessed a huge development in the research on natural products from marine organisms, especially from marine microbes [1][2][3][4]. Due to their diverse structures and fascinating activities, metabolites from marine-derived fungi have attracted the attention of chemists and biologists [5][6][7]. According to our statistics, nearly two thousand compounds have been isolated from marine-derived fungi since 1945, many of which exhibit potent biological activities [8,9]. During the last decade, we have chemically investigated marine-derived fungal species isolated from many different marine habitats and discovered a variety of structurally interesting natural products with various biological activities, such as sclerotides A and B [10], cottoquinazolines B-D [11], penipenes A-F [12], and phomazines A-C [13]. As part of our studies to search for new bioactive compounds from marine-derived fungi, our attention was drawn to an endozoic fungal strain, Cladosporium sp. OUCMDZ-1635, isolated from a sponge ( Figure S26). The ethyl acetate (EtOAc) extract of the fermentation broth was found to show moderate cytotoxicity against cancer cell lines at 100 µg/mL. Furthermore, HPLC-UV analysis of the EtOAc extract revealed one main peak with UV absorptions at 278 nm and 322 nm. Chemical investigation on this strain has resulted in the isolation of a new hybrid polyketide, cladodionen (1), and a new analogue of abscisic acid, which we named cladosacid (2). Cladodionen (1) was isolated as an inseparable mixture of two geometric isomers because of dynamic interconversion (Figure 1). Compound 1 represents a new natural skeleton and structurally belonged to hybrid polyketides, and only four examples, bripiodionen [14], apiodionen [15], vermelhotin [16] and hypoxyvermelhotins [17], have the similar skeleton ( Figure S1).

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter, and UV spectra were recorded on a Beckman DU 640 spectrophotometer. ECD data were collected using a JASCO J-715 spectropolarimeter. IR spectra were obtained on a Nicolet Nexus 470 spectrophotometer as KBr discs. NMR spectra were recorded using a JEOL JNM-ECP 600 spectrometer or an Agilent 500 MHz DD2 spectrometer using TMS as an internal standard or residual solvent signals for referencing. HRESIMS spectra were determined using a Micromass ® Q-TOF Ultima Global GAA076 LC mass spectrometer (Waters Aisa, Ltd. Singapore). Semi-preparative HPLC was carried out using an ODS column (YMCpack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min). Thin layer chromatography (TLC) and column

The Bioactivities of Compounds 1-3 from Cladosporium sp. OUCMDZ-1635.
Compounds 1 and 2 were tested for cytotoxic activities against human lung carcinoma cell line

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter, and UV spectra were recorded on a Beckman DU 640 spectrophotometer. ECD data were collected using a JASCO J-715 spectropolarimeter. IR spectra were obtained on a Nicolet Nexus 470 spectrophotometer as KBr discs. NMR spectra were recorded using a JEOL JNM-ECP 600 spectrometer or an Agilent 500 MHz DD2 spectrometer using TMS as an internal standard or residual solvent signals for referencing. HRESIMS spectra were determined using a Micromass ® Q-TOF Ultima Global GAA076 LC mass spectrometer (Waters Aisa, Ltd. Singapore). Semi-preparative HPLC was carried out using an ODS column (YMC-

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter, and UV spectra were recorded on a Beckman DU 640 spectrophotometer. ECD data were collected using a JASCO J-715 spectropolarimeter. IR spectra were obtained on a Nicolet Nexus 470 spectrophotometer as KBr discs. MR spectra were recorded using a JEOL JNM-ECP 600 spectrometer or an Agilent 500 MHz DD2 spectrometer using TMS as an internal standard or residual solvent signals for referencing. HRESIMS spectra were determined using a Micromass ® Q-TOF Ultima Global GAA076 LC mass spectrometer (Waters Aisa, Ltd. Singapore). Semi-preparative HPLC was carried out using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 µm, 4 mL/min). Thin layer chromatography (TLC) and column chromatography (CC) were performed on plates pre-coated with silica gel GF254 (10-40 µm, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Buckinghamshire, England), respectively. Vacuum-liquid chromatography (VLC) utilized silica gel H (Qingdao Marine Chemical Factory).

Collection and Phylogenetic Analysis of Strain OUCMDZ-1635
The fungus OUCMDZ-1635 was isolated from a sponge sample ( Figure S26) collected from Xisha Islands of China in August 2010. After it was ground into powder, the sample (1.0 g) was diluted to 10 −2 g/mL with sterile water, 100 µL of which was deposited on a PDA (200 g potato, 20 g glucose, 20 g agar per liter of sea water) plate containing chloramphenicol (100 µg/mL) as a bacterial inhibitor. A single colony was transferred onto another PDA plate and was identified as Cladosporium sp. according to its morphological characteristics and ITS gene sequences (GenBank accession No. KT336457). A reference culture of OUCMDZ-1635 is maintained at −80 • C and deposited in W. Zhu's laboratory.

Cultivation and Extraction of OUCMDZ-1635
Fungal strain OUCMDZ-1635 was cultured on slants of PDA medium at 28 • C for 5 days. Plugs of agar supporting mycelium growth were cut and transferred aseptically to 20 × 1000 mL Erlenmeyer flasks each containing rice medium composed of 80 g rice and 120 mL seawater. The flask was incubated at room temperature under static conditions for 30 days. The cultures were extracted three times by EtOAc (each 300 mL) and the combined EtOAc extracts were dried in vacuo to yield 6.5 g of extract.

Preparation of 3
To a stirred suspension of sodium hydride (6.0 mg, mixture of 60% NaH in mineral oil) in N,Ndimethylformamide (DMF) (1.0 mL) was added a solution of compound 1 (5.0 mg) in DMF (1.0 mL) at −5 • C. After stirring for 0.5 h, 15 µL of CH 3 I was added dropwise and the mixture was stirred for another 0.5 h. Then the reaction was quenched by the addition of saturated aqueous NH 4 Cl solution (2.0 mL) and extracted with CH 2 Cl 2 (3 × 10 mL). The organic layers were combined and washed with H 2 O (2 × 10 mL) and concentrated in vacuum. The residue was then purified by HPLC over an ODS column (70% MeOH/H 2 O) to afford compound 3 (4.5 mg, t R = 6.45 min, 80% yield).