Prenylated Coumarins from Heracleum stenopterum, Peucedanum praeruptorum, Clausena lansium, and Murraya paniculata

Four hitherto unknown prenylated coumarins, namely 6″-O-β-d-apiofuranosylapterin (1), 4′-O-isobutyroylpeguangxienin (2), 6-(3-methyl-2-oxobutyroyl)-7-methoxycoumarin (3), and 6-hydroxycoumurrayin (4), were isolated from the ethanol extract of Heracleum stenopterum, Peucedanum praeruptorum, Clausena lansium, and Murraya paniculata, respectively. Their chemical structures were established on the basis of extensive spectroscopic analysis. Compound 2 exhibited in vitro cytotoxic activity against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW-480) with IC50 values ranging from 15.9 to 23.2 μM.

Compound 3, yellow amorphous powder, possessed a molecular formula of C 15 [16]. This unit was attached to C-6 from the HMBC correlation of H-5 to C-1 0 , while the methoxy at C-7 by a weak but clear 4 J correlation from the methoxy protons to C-8. Accordingly, compound 3 was determined as 6-(3-methyl-2-oxobutyroyl)-7-methoxycoumarin.
Compound 4, white amorphous powder, had a molecular formula of C 16   . All of the above spectroscopic data were generally consistent with those of 6-methoxycoumurrayin [16], except that a methoxy group was replaced by a hydroxy group. The two methoxy groups were located at C-5 and C-7 on the basis of the HMBC correlations from H-4 to C-5, 5-OCH 3 to C-5, H-1 0 to C-7/C-8a, and from 7-OCH 3 to C-7. Thus, compound 4 was identified as 6-hydroxycoumurrayin.
The in vitro cytotoxicity of these new coumarins (1-4) was evaluated against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW-480) using the MTS method. DDP (cisplatin) and paclitaxel were used as positive controls. Compound 2 exhibited cytotoxic activity with IC 50 values ranging from 15.9 to 23.2 lM for all tested cell lines, while the other compounds were inactive (IC 50 values [40 lM).

General Experimental Procedures
Optical rotations were measured on a Jasco P-1020 automatic digital polarimeter. UV data were obtained from HPLC online analysis. NMR spectra were carried out on a Bruker AV-400, DRX-500, Avance III 600, or AV-800 spectrometer with deuterated solvent signals used as internal standards. ESI and HRESIMS were performed with a Shimadzu LC-IT-TOF mass spectrometer equipped with an ESI interface (Shimadzu, Kyoto, Japan). Silica gel 200-300 Fractions were monitored and analyzed by TLC, in combination with an Agilent 1200 series HPLC system equipped by an Extend-C18 column (5 lm, 4.6 9 150 mm). Table 4

6-(3-Methyl-2-oxobutyroyl)-7-methoxycoumarin (3)
Yellow amorphous powder; UV (MeOH) k max : 215 (sh), 227 (sh), 261, 308, 340 nm; 1 H NMR and 13 C NMR data: see Table 3    . Briefly, 100 lL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 9 10 5 cells/mL in 100 lL medium. Each cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel as positive controls. After the incubation, 20 lL MTS and 100 lL medium was added to each well after removal of 100 lL medium, and the incubation continued for 2-4 h at 37°C. The optical density was measured at 492 nm using a Multiskan FC plate reader (Thermo Scientific, USA). The IC 50 value of each compound was calculated according to the Reed and Muench method.