New Cytotoxic Azaphilones from Monascus purpureus-Fermented Rice (Red Yeast Rice)

Using a cell-based cytotoxicity assay three new cytotoxic azaphilones, including two stereoisomers and designated monapurones A-C (1-3), were isolated from the extract of Monascus purpureus-fermented rice (red yeast rice). Their structures were elucidated by detailed interpretation of spectroscopic and chemical data. The relative configurations were assigned on the basis of analysis of NOE data, and the absolute configurations were determined by direct comparison of their CD spectra with those of known azaphilones and chemical correlations. In the in vitro assays, monapurones A-C (1-3) showed selective cytotoxicity against human cancer cell line A549 with IC50 values of 3.8, 2.8 and 2.4 μM respectively, while exhibiting no significant toxicity to normal MRC-5 and WI-38 cells at the same concentration.


Introduction
The fungus Monascus has traditionally been used to prepare red fermented rice, as a natural food colorant or food preservative of meat and used as a folk medicine in east Asia for centuries [1,2], Many reports indicated that metabolic products from fermentation of Monascus species were utilized to decrease blood pressure, lower plasma cholesterol levels and blood sugar. They also showed antitumor, antibacterial and antioxidative activities [3][4][5][6][7][8]. As part of a program to search for potential antitumor agents from natural sources, we investigated the extract of red yeast rice fermented with Monascus purpureus. From the petroleum ether-soluble portion of the EtOH extract of this material, three new cytotoxic azaphilones, including two stereoisomers, and designated monapurones A-C (1-3), were isolated. In the in vitro assays, monapurones A-C (1-3) showed selective cytotoxicity against human cancer cell line A549 with IC 50 value of 3.8, 2.8 and 2.4 M respectively, while exhibiting no significant toxicity to normal MRC-5 and WI-38 cells at the same concentration. To our knowledge, this is the first isolation of azaphilones with a C 20 skeleton. Herein, we report the isolation, characterization and biological activity of monapurones A-C (1-3).

Results and Discussion
The petroleum ether portion with the highest cytotoxic activity ( IC 50 < 50 μg/mL ) was subjected to column chromatography over silica gel, Sephadex LH-20 and reverse-phase HPLC to yield three new azaphilones: monapurones A-C (1-3, Figure 1) Monapurones A (1) was obtained as a yellow oil. Its IR absorptions at 3,403, 1,712, and 1,618 cm -1 implied the existence of hydroxyl, carbonyl, and double bond functional groups, respectively. The UV spectrum showed absorption maxima at 354, 284 and 230 nm, indicating the presence of an extended conjugated system. High-resolution mass spectral analysis suggested a molecular formula C 20 H 26 O 4 (m/z 330.1827 [M] + ) with eight degrees of unsaturation, which was consistent with the structural information provided by 1 H-NMR and 13 C-NMR spectra.
The 13 C-NMR spectrum of 1 showed 20 carbons, and the DEPT experiment differentiated them to be 3  CH 3 , 5  CH 2 , 6  CH, and 6  C ( Table 2). On the basis of the chemical shift values, 10 of them were assigned to be sp 2 carbons including two carbonyls (δ c 198.3, 209.8) and eight olefinic carbons, and 10 of them were sp 3 carbons (one oxygen-bearing, δ > 70 ppm). The structure of 1 was finally elucidated as azaphilone by analysis of the data obtained in 2D NMR experiments and comparison with those of previously characterized azaphilones [9][10][11][12].
The proton and protonated carbon signals in the NMR spectra of 1 were assigned unequivocally on the basis of heteronuclear correlations in the HMQC spectrum. In the HMBC spectrum of 1, a series of two-and three-bond correlations from H-1 to C-3, C-8, C-4a, and C-8a, from H-4 to C-3, C-5, C-4a and C-8a, from H-5 to C-4, C-6, C-7, C-4a and C-8a, from CH 3 -9 to C-6, C-7 and C-8 ( Figure 2, arrows), in combination with their chemical shift values, revealed unequivocally a 7-hydroxy-7methyl-7,8-dihydro-6H-isochromen-6-one nucleus (azaphilone skeleton) for 1. The presence of prop-1-enyl unit was established by 1 H-1 H coupling between H-11/H-10 and CH 3 -12 and long-range correlations between CH 3 -12 and C-10 and C-11. In addition, HMBC correlations from H-4 to C-10, from H-10 to C-3 and C-4 and from H-11 to C-3 allowed the prop-1-enyl unit to be connected to C-3 of the azaphilone nucleus. Futher investigation of 1 H-1 H COSY and HMBC spectra of 1 ( Figure 2) revealed the presence of the 2-oxoheptyl side chain, which was located at C-8 of the azaphilone nucleus based on the homonuclear coupling correlation between H-8 and H 2 -13 and the long-range correlations from H-8 to C-13 and C-14, and from H 2 -13 to C-7, C-8 and C-8a. Thus, the full planar structure of 1 was assigned as shown in Figure 1. The relative stereochemistry of 1 was verified by a careful analysis of its 600 MHz NOESY (in CD 3 COCD 3 ) and 500 MHz NOESY (in CDCl 3 ) spectroscopic data. NOE correlations between H-8 and CH 3 -9 revealed that these protons were cofacial and arbitrarily defined as having an α-orientation. The 2-oxoheptyl side chain and OH-7 were assigned to be β-configuration judging from the NOE correlations between OH-7 and H-13β ( Figure 3). The C-10-C-11 double bond was established to be E-form by the coupling constant (Table 1) and the NOE correlation between H-10 and CH 3 -12.
The NMR spectral data of 2 were similar to those of 1, except that the carbonyl signal(δ c 209.8, C-14) in 1 was replaced by a hemiacetal carbon signal (δ c 108.0, C-14) in 2 and C-7 of 2 was downfield shifted by Δδ c 9.6 ppm compared to that of 1. These data indicated the linkage between C-7 and C-14 via oxygen atom to form an five-membered ring in 2, which was further confirmed by the 2D NMR experiments and the absence of hydroxyl absorption band at 3,403 cm -1 in the IR spectrum. Meanwhile, the NMR spectra of 2 showed the presence of an extra methoxyl signal (δ H 3.16, δ c 48.5), which was located at C-14 on the basis of the HMBC correlation from the methoxy protons at C-14 to C-14.
To verify the deduced stereochemistry at C-7 of 2 and 3, the two compounds were subjected to acid hydrolysis with 0.1N HCl at room temperature. Almost all 2 and 3 were converted into 1 after two hours. The products in both reactions were identified to be 1 based on the identical 1 H-NMR, 1 H-1 H COSY, NOESY and CD data. On the basis of the above results, the chiral centers at C-7 of 2 and 3 were further established as R configuration.
In the in vitro cytotoxic assays, monapurones A-C (1-3) showed potent selective cytotoxicity against human cancer cell line A549 with IC 50 value of 3.8, 2.8 and 2.4 M respectively, while posing no toxicity to normal MRC-5 and WI-38 cells at the same concentration.

General
CD spectra were recorded on a JASCO J-810 circular dichroism spectrometer. UV data were determined on a Hitachi UV-3000 spectrometer, and IR spectra were recorded as KBr disks on a Nicolet Impact 400 FT-IR Spectrophotometer. 1D-and 2D-NMR spectra were acquired in CDCl 3 and CD 3 COCD 3 with TMS as internal standard on Varian 500 or 600 MHz spectrometers. Mass spectra including high-resolution mass spectra were recorded on a JEOL JMS AX-500 spectrometer. Column chromatography was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Inc. China), RP-18 reverse phase silica gel (43-60 m) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala Sweden). LPLC separation was performed with Combiflash (ISCO Companion). HPLC was performed with a Waters 600 system with 2996 DAD-detector, with an Alltima (C18, 250  10 mm i.d., 5 μm) column. TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 8 % H 2 SO 4 in 95 % EtOH followed by heating.

Fungal material
Red yeast rice was prepared from cooked paddy rice inoculated with Monascus purpureus B0708, purchased from the Beijing Dawn Aerospace Bio-tech Company. The sample was deposited at Beijing Union University, Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing 100083, China.

Extraction and isolation
The dried red yeast rice powder (4.5 kg) was extracted with 50 L of 95 % EtOH, 80 % EtOH and 60 % EtOH, respectively, at room temperature (×3, each for 3 hours). After removal of the solvent under reduced pressure, the residue (850.0 g) was suspended in 3 L of H 2 O, and then partitioned sequentially with petroleum ether (3 times with 5 L each) and EtOAc (4 times with 5 L each) to yield a petroleum ether (38.0 g), a EtOAc (223.5 g) and a H 2 O (588.5 g) fractions, respectively.

Acid hydrolysis of 2 and 3
Compound 2 (5.0 mg in MeOH) was hydrolyzed with 0.1 N HCl (0.5 mL) at room temperature for 2 hours. The reaction progress was monitored by TLC eluted with CHCl 3 -CH 3 COCH 3 (6:1). After evaporation of the solvent under a stream of nitrogen, the residue dissolved in CD 3 COCD 3 with TMS as internal standard was analyzed by Varian 600 MHz spectrometer. Then, the solvent was removed under a nitrogen stream and the residue dissolved in MeOH was analyzed on the JASCO J-810 circular dichroism spectrometer. Compound 3 (5.0 mg) was hydrolyzed using the same method.

In vitro cytotoxicity assay
WI-38, MRC-5, HCT-8, Bel7402, BGC-823, A549, MCF-7, and A2780 cells were seeded in 96-well microtiter plates at 1,200 cells/well. After 24 h, the tested compounds were added to the systems. After 96 h of drug treatment, cell viability was determined by measuring the metabolic conversion of MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) into purple formazan crystals by active cells. MTT assay results were read using a MK 3 Wellscan (Labsystem Drogon) plate reader at 570 nm. All compounds were tested in five concentrations and were dissolved in 0.1 % DMSO each well. Each concentration of the compounds was tested in three parallel wells. IC 50 values were calculated using Microsoft Excel software.  Tables 1 and 2.