Bioactive Phenylpropanoids from Daphne feddei

A investigação de extrato acetônico de folhas e caules de Daphne feddei forneceu três novos fenilpropanóides, fedeicetonas A-C (1-3), juntamente com nove fenilpropanóides conhecidos (4-11). Suas estruturas foram elucidadas com base em métodos espectroscópicos. Os compostos 1-3 foram testados com relação as suas atividades anti-HIV-1 e citotoxidades frente a células cancerígenas. Os resultados mostraram que as atividades citotóxicas e anti-HIV-1 dos compostos 1-3 são modestas.


Introduction
The genus Daphne (Thymelaeaceae) comprises about 70 species, 35 of which grow in China.Daphne feddei Levl., which is known as "Dian Rui Xiang" (Chinese), is distributed in Yunnan, Sichuan and Guizhou Provinces of China.It is a kind of evergreen shrub.Its leaves and stems have been used as folk medicine to treat the injuries from falls and bruises. 1 Previous chemical studies on D. feddei Levl.][11] Our previous studies on Daphne feddei collected from Dali Prefecture of Yunnan Province resulted in the isolation of several lignans and phenylpropanoids including two new secolignans and one new neolignan. 12As a continuation of investigation on chemical constituents of the same Daphne feddei plant, three new phenylpropanoids (1-3), together with nine known phenylpropanoids (4-12), were isolated.In addition, the anti-HIV-1 and cytotoxic activities of compounds 1-3 were evaluated.This article deals with the isolation, structural elucidation and biological activities of the new compounds.

Results and Discussion
The 70% aq.acetone extract of D. feddei was suspended in H 2 O and partitioned with ethyl acetate.The ethyl acetate partition was repeatedly subjected to column chromatography over Si gel and preparative HPLC (high performance liquid chromatography) to afford compounds 1-12, including three new phenylpropanoids, named feddeiketones A-C (1-3), together with nine known phenylpropanoids, p-coumaric acid (4), 13 caffeic acid (5), 14 ferulic acid (6), 15 coniferoside (7), 16 isoconiferin (8), 17 turbinataphenol A (9), 18 nervolan B (10), 19 nervolan C (11) 20 and 1,2-O-dicaffeoylcyclopenta-3-ol (12). 21The structures of compounds 1-12 are shown in Figure 1.In addition to three new compounds, compounds  10.98).Strong absorption bands accounting for hydroxyl (3382 cm -1 ), carbonyl (1715, 1705 cm -1 ) and aromatic group (1636, 1508, 1452 cm -1 ) could also be observed in its IR (infrared) spectrum.The UV spectrum of 1 showed absorption Table 1. 1 H and 13 C NMR data (in Therefore, the structure of 1 was elucidated as that shown in Figure 1, and it has been trivially named as feddeiketone A. Compound 2, that was also obtained as pale yellow gum, has a molecular formula C 11 H 14 O, based on the HRESIMS showing a sodiated molecular ion at m/z 249.0731 [M + Na] + (calc.m/z 249.0739).The 1 H and 13 C NMR spectra of 2 were very similar to those of 1.The IR and UV spectra were also similar to those of compound 1.The differences from 1 were the absence of the acetoxy group signal and the appearance of the hydroxyl proton signal (d H 4.99 brs) in 2. Therefore, the structure of 2 was established and was named feddeiketone B.
Compound 3 was also obtained as pale yellow gum.The 1 H and 13 C NMR spectra of 3 were very similar to those of 2. The obvious shift differences resulted from the substituent group variations in the aromatic ring.For compound 3, the HMBC spectrum showed that a methoxyl group was located at C-3, and a methylenedioxy group at C-4 and C-5.Accordingly, the structure of 3 was determined as shown and named feddeiketone C.
The cytotoxicity tests for the isolates were performed using a previously reported procedure. 22All treatments were performed in triplicate.In the MTT assay, the IC 50 was defined as the concentration of the test compound resulting in a 50% reduction of absorbance compared with untreated cells.The cytotoxic abilities of compound 1-3 against HL-60, Hep-G2, KB and MDA-MB-231 tumor cell lines were performed using the MTT-assay (with doxorubicin as the positive control).The results were shown in Table 2 and showed that compounds 1-3 have significant potential cytotoxic activities.

Conclusions
The spectroscopic analyses of compounds isolated from leaves and stems of Daphne feddei.revealed three new phenylpropanoids (named feddeiketones A-C (1-3)), together with nine known phenylpropanoids (4-11).These new compounds had their cytotoxic activities and anti-HIV-1 bioactivity studied, with modest activities.

Experimental
General procedures IR spectra were measured on a Bruker Tensor 27 spectrometer with KBr pellets.UV spectra were measured on a Shimadzu UV-2401A spectrophotometer.Optical rotations were measured on a Horiba SEPA-300 polarimeter. 1 H and 13 C NMR and 2D NMR spectra were recorded on Bruker DRX-500, with chemical shifts (d) in ppm relative to tetramethylsilane (TMS) as internal standard and coupling constants in Hz.HRESIMS data were measured

Plant material
The leaves and stems of D. feddei were collected from Dali Prefecture, Yunnan Province, People's Republic of China, in September 2009, and authenticated by Professor Chen Y. J. (Yunnan University of Nationalities).A voucher specimen (YNNI 09-9-12) is deposited the Herbarium of Key Laboratory of Ethnic Medicine Resource Chemistry, Yunnan University of Nationalities.

Anti-HIV-1 assay
Cytophatic effect inhibitory assay using C8166 cells infected with HIV-1 was used to evaluate anti-HIV-1 activity (EC 50 ).The cytotoxicities of compounds 1-3 against C8166 cells (CC 50 ) were also evaluated using the MTT method and allowed the determination of therapeutic index (TI). 21

Cytotoxicity assay
Cytotoxic activities of compounds 1-3 against cancer cell lines HL-60, Hep-G2, KB and MDA-MB-231 were measured by the MTT method using doxorubicin as the positive control. 22ddeiketone A ( 1

13
C NMR, DEPT and 1 H NMR spectra of feddeiketones A-C, HSQC, HMBC, HRESIMS spectra of feddeiketone A, are available free of charge at http://jbcs.sbq.org.br as PDF file.

8, 9, 10, 11 and 12 were
isolated from the plant of Daphne genus for the first time.Vol.23,No. 4, 2012Compound 1 was isolated as pale yellow gum.