Two New Derivatives of 2, 5-Dihydroxyphenylacetic Acid from the Kernel of Entada phaseoloides

Two new aromatic compounds, butyl 2,5-dihydroxyphenylacetate (1) and butyl 2-O-β-D-glucopyranosyloxy-5-dihydroxyphenylacetate (2), together with three known ones, methyl 2,5-dihydroxyphenylacetate (3), ethyl 2,5-dihydroxyphenylacetate (4) and 2-O-β-D-glucopyranosyloxy-5-hydroxyphenylacetic acid (5), were isolated from the EtOH extract of the kernel of Entada phaseoloides. The structures of the new compounds were elucidated by MS and NMR experiments. Compounds 1, 3 and 4 displayed potent inhibitory activities against HIV-1 replication, with EC50 values of 9.80 μM, 11.70 μM and 9.93 μM, respectively.


Introduction
Entada phaseoloides (L.) Merr. is the sole species of Entada genus (Leguminosae), widely distributed in south China, esp. in Yunnan and Hainan provinces. The kernel of E. phaseoloides has been commonly used as an herbal medicine by the Dai nationality for the treatment of hemostasis and OPEN ACCESS detoxification [1]. Some investigations suggested that it had antidiabetic [2], anti-inflammatory [3], and molluscicidal activities [4]. In 1955, Barua first obtained a triterpene acid from its kernel [5]. After that, more compounds from this plant were reported, such as phenylacetic acid esters [6,7], triterpene saponins [8], phenolic acids [9,10], chalcone glycosides [11] and sulfur-containing amides [12,13]. As one of the components in Qi-wei Ke-Teng-Zi Wan [14], a famous formula of medicines used in the Dai nationality, the active constituents of the kernel of E. phaseoloides are still unknown. Herein we report the isolation, structure elucidation, and anti-HIV activity of five aromatic compounds from the kernel of Entada phaseoloides.

Results and Discussion
The EtOH extract from air-dried kernels (7 kg) of E. phaseoloides was extracted with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract was separated by repeated column chromatography to give five aromatic compounds ( Figure 1) including two new ones (compounds 1, 2). The known compounds were readily identified as methyl 2,5-dihydroxyphenyl acetate (3)     Compound 1 was obtained as a white crystalline solid. The molecular formula was determined as C 12 H 16 O 4 (five degrees of unsaturation) on the basis of its TOF-ESI-MS at m/z 247.0963 [M+Na] + . The IR spectrum of 1 showed absorption bands at 3390 (OH), 1722 (C=O) cm −1 . The 1 H-NMR data ( Table 1) of 1 revealed similar structural features as those of 3, except for the additional signals of the butyl group at δ H 0.92 (3H, t, J = 7.5 Hz), 1.64 (2H, m), 1.37 (2H, m), 4.14 (2H, t, J = 6.5 Hz). This implied that the methyl in 3 was replaced by the butyl in compound 1. HMBC correlation of H-9 with C-8 showed the butyl group was connected to the ester bond. Hence, 1 was elucidated as butyl 2,5-dihydroxyphenyl acetate.
Compound 2 was obtained as a white crystalline solid and displayed similar UV and IR profiles to those of 1. The molecular formula was determined to be C 18 H 26   All isolated compounds were evaluated for anti-HIV activity against VSVG/HIV pseudotyped virus using zidovudine as the positive control. Compounds 1, 3 and 4 exhibited inhibitory activities against HIV-1 replication with EC 50 values of 9.80 μM, 11.70 μM and 9.93 μM, respectively, while the EC 50 values of zidovudine was 11.70 nM. However, compounds 2 and 5 did not show inhibition at the concentration of 10 μM.

General
NMR spectra were measured on a Bruker AM 500 NMR spectrometer as the internal reference and chemical shifts are expressed in ppm. TOF-ESI-MS spectra were measured on a Waters Synapt G2 mass spectrometer. EIMS data were recorded on a Zabspec E mass spectrometer. IR spectra were recorded on a Shimadzu FTIR-8400S spectrophotometer. UV spectra were run on a Shimadzu UV-2550 UV/Vis spectrophotometer. TLC was performed on silica gel GF254 (10-40 μm; Qingdao Marine Chemical, Inc., Qingdao, China). Column chromatography was performed on silica gel (100-200 or 200-300 mesh; Qingdao Marine Chemical, Inc.).

Plant Material
The kernal of Entada phaseoloides was collected from Xishuangbanna, Yunnan province, in January, 2010. The sample was identified by one of the authors C. Z. Peng, and a voucher specimen (No. 20100128) has been deposited in the herbarium of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College.