Triterpenoid alkaloid derivatives from Buxus rugulosa

Four new triterpenoid alkaloid derivatives, buxrugulines A–D (1–4), together with four known ones (5–8), were isolated from the leaves and stems of Buxus rugulosa. The structures of compounds 1–4 were elucidated by NMR and MS spectroscopic analysis. All compounds were assayed for their cytotoxicities against HL-60, SMMC-7721, A549, MCF-7, and SW480 cells lines.


Introduction
Buxus rugulosa, belonging to the Buxus genus of the family Buxaceae, is a dwarf shrub growing in the rocky mountains in the northwest district of Yunnan Province. In previous phytochemical investigations of the genus Buxus, more than 220 triterpenoid alkaloid derivatives have been isolated 1,2 . This type of alkaloid showed interesting pharmacological activities such as anti-myocardial ischemia 3,4 , antibacterial activities 5,6 , and inhibition of cholinesterases [7][8][9] . In our previous studies from Buxus plants, new alkaloids with diverse structures and promising cytotoxic activities have been reported 10,11 . As part of this study, we have examined the stems and leaves of B. rugulosa, and consequently isolated four new triterpenoid alkaloid derivatives, buxrugulines A-D (1)(2)(3)(4), along with four known ones, N 20 -acetoxy-cyclovirobuxin D (5) 12 , (+)-16αacetoxybuxabenzamidienine (6) 13,14 , moenjodaramine (7) 9,15,16 , irehine (8) 17 . Herein we report the isolation and structural elucidation of the new compounds, as well as cytotoxic activities of the isolates from B. rugulosa.

Results and Discussion
A crude alkaloid fraction of B. rugulosa yielded eight triterpenoid alkaloid derivatives by repeated silica gel, amino silica gel, C-18 and Sephadex LH-20 chromatography.  *To whom correspondence should be addressed. E-mail: mhchiu@mail.kib.ac.cn 129.7), five methylenes, and six methyl groups. Comparison of the spectroscopic data of 1 and cyclobuxotriene 13 revealed similarities except for the absence of a methyl on the nitrogen at C-20 and the presence of a double bond at C-6/7 in 1. This was supported by the HMBC correlations of H-5 (δ H 2.73) with C-6 (δc 128.4) and of H-8 (δ H 1.91) with C-7 (δc 127.6), C-9 (δc 146.1) ( Figure 2). Therefore, 1 was elucidated as shown, and named buxruguline A. ]. All the data indicated that compound 2 was similar to buxpiine 7 , and the distinct difference between them was that a oxygenated methine (δ C ≈ 72) of C-16 in buxpiine was replaced by a methylene (δ C 34.2) in 2. This deduction was supported by HMBC correlations from H-21 (δ H 1.08) to C-20 (δ C 212.9), C-16 (δ C 34.2) and from H-17 (δ H 2.66) to C-20, C-16 and C-13 (δ C 42.7). H-5 is invariably α-oriented in this type alkaloid 18,19 , the ROESY correlation of H-3 (δ H 2.85) with H-5 (δ H 2.12) indicating an α-orientation of H-3 and βorientation of the amino functionality. So, the structure of 2 was elucidated as shown in Figure 1.
Buxruguline C (3)  The moleculr formula of buxruguline D (4) was assigned as C 29 H 48 N 2 O 3 on the basis of the NMR data (Table 1) and HRESIMS. Comparison of the spectroscopic data of 4 and 3 revealed similarities cycloartane-type triterpenoid skeleton. The notable difference was that a OH functionality at C-16 in 3 was replaced by acetoxy group in 4, which confirmed by the downfielded H-16 (δ H 4.11) proton signal and the HMBC correlation from H-16 to the O-acetyl carbonyl carbon at δ C 169.3 (C). Moreover 4 has one less hydroxyl function at C-30 and one less methyl group on the nitrogen at C-3 than 3. Consequently, compound 4 was elucidated as shown and has been accorded the trivial name buxruguline D.

Experimental Section
General Experimental Procedures. Melting points were determined on a YU-HUA X-4 melting point apparatus. Optical rotations were obtained with a Horiba SEAP-300 polarimeter. Infrared spectra were recorded on a Shimadzu IR-450 instrument by using KBr pellets. NMR spectra were measured on a Bruker AV-400 and DRX-500 instrument (Bruker, Zűrich, Switzerland) with TMS as internal standard. HR-ESIMS data were recorded on a VG Auto Spec-3000 spectrometer.  ent, and was further repeatedly separated on amino silica gel column chromatography, eluted with CHCl 3 -MeOH (20:1, 10:1), to give 2 (6 mg), 3 (12 mg), 6 (76 mg). Cell Culture and Cytotoxicity Assay. A panel of human tumor cell lines was used: promyelocytic leukemia HL-60, hepatocellular carcinoma SMMC-7721, alveolar basal epithelial carcinoma A549, breast adenocarcinoma MCF-7, and colon cancer SW480. The cells lines were obtained from the Shanghai cell bank of China. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA) at 37°C in a humidified atmosphere with 5% CO 2 .
Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in the living cells with the MTT (MTT, sigma, USA) method described before 21 , and using cisplatin (DDP, sigma, USA) as control. Cell growth inhibition curve was graphed and the IC 50 value of each compound was calculated by the Reed and Muench method 22 .