Woodylides A–C, New Cytotoxic Linear Polyketides from the South China Sea Sponge Plakortis simplex

Three new polyketides, woodylides A–C (1–3), were isolated from the ethanol extract of the South China Sea sponge Plakortis simplex. The structures were elucidated by spectroscopic data (IR, 1D and 2D NMR, and HRESIMS). The absolute configurations at C-3 of 1 and 3 were determined by the modified Mosher’s method. Antifungal, cytotoxic, and PTP1B inhibitory activities of these polyketides were evaluated. Compounds 1 and 3 showed antifungal activity against fungi Cryptococcus neoformans with IC50 values of 3.67 and 10.85 µg/mL, respectively. In the cytotoxicity test, compound 1 exhibited a moderate effect against the HeLa cell line with an IC50 value of 11.2 µg/mL, and compound 3 showed cytotoxic activity against the HCT-116 human colon tumor cell line and PTP1B inhibitory activity with IC50 values of 9.4 and 4.7 µg/mL, respectively.

Marine sponges provide a wide range of polyketides with antibacterial [4], antiviral [5], antitumor [6], antimalarial [7], and taxol-like microtubule-stabilizing activities [8]. The sponge-derived polyketides often contain cyclic peroxides and lactone functionalities, linear and bicyclic carbon frameworks [1,9], and macrolide and aromatic groups in some cases [8,10]. A prolific source of new and bioactive polyketides derived from sponges of the genus Plakortis attracted our attention. As part of our ongoing search for new pharmacologically active lead compounds from the marine sponges collected off Xisha Islands in the South China Sea [11,12], we investigated polyketides from the marine sponge Plakortis simplex. A preliminary study led to the isolation of two new polyketides named simplextones A and B with an unusual cyclopentane skeleton [6]. The interesting chemical and bioactive significance of P. simplex prompted us to continue the study of this sponge, which has led to the isolation of three new linear polyketides, named as woodylide A (1), B (2) and C (3) (Figure 1) [13], which are the acyclic diol analogues of the cyclic polyketide peroxides isolated from the genus of Plakortis [14,15]. This article describes the isolation, identification and bioactivity of the new compounds.
To confirm if compound 1 could be an artifact formed from 3 during the isolation processes, a solution of 3 was kept at room temperature for three days in the presence of Si-60 or RP-18 gel in MeOH, respectively. The formation of 1 was not observed, thus suggesting that compound 1 may be a natural product and not an artifact.

General Experimental Procedures
Optical rotations were determined with a Perkin-Elmer 341 polarimeter with 1 mm cell. IR spectra were recorded on a Bruker vector 22 spectrometer with KBr pellets. The NMR experiments were conducted on Bruker AVANCE-600 and Bruker AMX-500 instruments. HRESIMS and ESIMS were obtained on a Q-Tof micro YA019 mass spectrometer. In antifungal evaluation, IC 50 values were calculated on XLfit 4.2 software (IDBS: Alameda, CA, USA, 2005). Reversed-phase HPLC was performed on YMC-Pack Pro C 18 RS (5 μm) columns with a Waters 1525/2998 liquid chromatograph. Column chromatographies were carried out on silica gel 60 (200-300 mesh; Yantai, China), Sephadex LH-20 (Amersham Biosciences). TLC was carried out using HSGF 254 plates and visualized by spraying with anisaldehyde-H 2 SO 4 reagent.

Antifungal Evaluation
Antifungal IC 50 values of woodylides A-C against C. neoformans were calculated as described by Ikhlas A. Khan et al. [20]. Amphotericin B was used as the positive control. Minimal Inhibition Concentration (MIC) values of woodylides A-C were determined against three indicators (C. albicans, T. rubrum, and M. gypseum), following the National Center for Clinical Laboratory Standards (NCCLS) methods [21,22]. Fluconazole was used as the positive control. Briefly, samples (dissolved in DMSO) were serially diluted in 20% DMSO/saline and transferred (10 µL)

Cytotoxicity Assay
The cytotoxicity of compounds 1-3 against HCT-116, A549, HeLa, QGY-7703, and MDA231 cell lines was evaluated by the MTT assay as described in a previous report [23]. Briefly, compounds were solubilized in DMSO with the working concentration of test substances ranging from 1 to 100 μg/mL. Cells at the exponential growth phase were harvested and seeded into 96-well plates. After incubation for 24 h, the cells were treated with various concentrations of test substances for 48 h and then incubated with 1 mg/mL MTT at 37 °C for 4 h, followed by dissolving in DMSO. The produced formazan was measured by the absorbance at 570 nm on a microplate reader. The IC 50 values were calculated on the basis of percentage inhibition using the linear regression method.

PTP1B Inhibitory Assay
PTP1B inhibitory activity was determined using a PTP1B inhibitory assay as described previously [24]. The enzymatic activities of the PTP1B catalytic domain were determined at 30 °C by monitoring the hydrolysis of pNPP. Dephosphorylation of pNPP generated the product pNP, which was monitored at an absorbance of 405 nm. In a typical 100 μL assay mixture containing 50 mmol/L 3-[N-morpholino]propanesulfonic acid (MOPs), pH 6.5, 2 mmol/L pNPP, and 30 nmol/L recombinant PTP1B, activities were continuously monitored and the initial rate of the hydrolysis was determined using the early linear region of the enzymatic reaction kinetic curve.

Conclusions
In this paper we report the isolation and the structural determination of three new linear polyketides, woodylides A-C, endowed with antifungal, antineoplastic, and PTP1B inhibitory activities, from the South China Sea marine sponge P. simplex. Unfortunately, due to the lack of compound 2, the absolute configuration at C-3 as well as the bioactivity of woodylide B could not be determined. Woodylide C exhibited a good PTP1B inhibitory activity, and deserves further study for its therapeutic potential against type II diabetes and obesity diseases.