Daphnane Diterpene Esters with Anti-proliferative Activities against Human Lung Cancer Cells from Daphne genkwa
Chemical and pharmaceutical bulletin
Daphne genkwa SIEB. et ZUCC. (Thymelaeaceae) is a traditional oriental medicine that is widely distributed in Korea and China. 1) The flower of this plant (Genkwa flos) has been traditionally used for abortifacient, diuretic, antitussive, expectorant, and anticancer effects. 1-3) Previous phytochemical investigations of Genkwa flos led to the isolation of various compounds such as flavonoids, coumarins, amides, and diterpenes. 4-9) Daphnane-type diterpenoids are typical constituents of plants
... ituents of plants from the families Thymelaeaceae and Euphorbiaceace. There have been reported several biological activities of daphnanes including antileukemia, piscicidal, toxicity, anticancer, abortion, and neurotrophy. 8,10,11) As part of our ongoing study for discovery of plant-derived anticancer agents, two new daphnane diterpenoids, namely, yuanhuahine (1) and yuanhualine (2), were isolated from Genkwa flos. The present study describes the structural elucidation of the two new isolates 1 and 2, along with their antiproliferative activities against human lung cancer cells and normal lung epithelial cells. Results and Discussion Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined as C 33 H 44 O 10 by the pseudo molecular ion peak at m/z 601.3013 [MϩH] ϩ in positive HR-FAB-MS. The IR spectrum of 1 showed absorptions at 3456 cm Ϫ1 for the hydroxyl groups and at 1695 cm Ϫ1 for the conjugated carbonyl group. The 13 C-and distortionless enhancement by polarization transfer (DEPT) 135 spectra of 1 indicated the presence of five methyls, seven methylenes, twelve methines, and nine quaternary carbons. In particular, an unusual quaternary carbon (C-1Ј) at d 117.2 was attributable to an orthoester group, which is a structural feature of daphnane-type diterpenoids. The quaternary carbon signals at d 209.7 and 173.4 in the 13 C-NMR spectrum were assignable to an a,b-unsaturated cyclopentanone and an ester group, respectively. The resonances of d H 3.56 and d C 64.3 (C-7) for an oxygenated methine group and at d C 60.7 (C-6) for an oxygenated quaternary carbon were upfield shifted, suggesting the presence of an epoxy group between C-6 and C-7. The 1 H-NMR spectrum displayed two olefinic proton signals at d H 5.01 and d H 4.95 assignable to the exomethylene group (C-16) and two proton signals at d H 3.94 and 3.81 attributed to an oxygenated methylene (CH 2 -20). All of these signals in the 1 H-and 13 C-NMR of 1 were similar to those of yuanhuadine (4), except for the ethyl signals (C-2Љ and C-3Љ) of 1 instead of the methyl signal for the acetyl group in 4. 8,    The methyl signal at d H 1.09 (3H, t, 7.5, H-3Љ) exhibited three-bond connectivity with the conjugated carbon at d C 173.4 (C-1Љ) in the heteronuclear multiple bond correlation (HMBC) spectrum of 1, indicating that the ethyl group was attached to the carbon at C-1Љ (Fig. 2) . The relative stereochemistry was determined by analysis of the rotating frame Overhauser enhancement spectroscopy (ROESY) Two new daphnane-type diterpene esters, yuanhuahine (1) and yuanhualine (2), were isolated from the flowers of Daphne genkwa (Thymelaeaceae) along with three known diterpene esters, yuanhuacine (3), yuanhuadine (4), and yuanhuagine (5). Their structures were determined by a combination of 1D and 2D NMR experiments, including correlation spectroscopy (COSY), heteronuclear multiple quantum correlation (HMQC), heteronuclear multiple bond correlation (HMBC), and rotating frame Overhauser enhancement spectroscopy (ROESY) sequences, and mass spectrometry. All the isolated compounds were tested against A549 human lung cancer cells and MRC-5 human normal lung epithelial cells. Compounds 1-5 exhibited potent anti-proliferative effects against A549 lung cancer cells with IC 50 values of 12-53 nM, whereas these compounds were relatively non-cytotoxic against MRC-5 normal lung epithelial cells.