Monoterpenoid indole alkaloids from Alstonia rostrata

Four new monoterpenoid indole alkaloids, alstrostines C-F together with thirteen known alkaloids were isolated from the leaves and twigs of Alstonia rostrata. All structures of new compounds were elucidated based on NMR, FTIR, UV, and MS spectroscopic data. Alstrostines C-E might originate from keto-enol tautomerism of preakummicine during biogenetic pathway of akummicine. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-012-0019-y and is accessible for authorized users.


Results and Discussion
The MeOH extract of A. rostrata leaves and twigs was partitioned between H 2 O and EtOAc after acid-alkali treating, and column chromatography was used to separate the    8 . The quaternary carbon signal at δ C 152.0 correlated with H-3 (δ H 3.75) and H-6 (δ H 1.65 and 2.82) in the HMBC spectrum was assigned to C-2. Detailed analysis of 13 C NMR and DEPT data revealed 1 might be belong to akuammicine-type alkaloids. Further NMR comparison with those of 12-methoxyechitamidine (7) 9 indicated that 1 was similar to 7 with exception for an additional methine (δ C 160.0 and δ H 9.12) in 1. The methine proton showed HMBC correlation to C-2, suggesting that the methine was connected with C-2 by an oxygen bridge with consideration of its molecular formula.
The UV spectrum of 2 and 3 also indicated the absorption bands of indole rings with a -anilineacrylate system together with FTIR spectrum. Hz, H-10). Its 13 C NMR and DEPT data showed similar pattern to 1 (see Table 1) except that the methine of C-19 was disappeared, instead a new signal of carbonyl group (δ C 207.2, s) was present in 2. Its HMBC correlations could support it, in which δ H 2.24 (H-18) was correlated with δ C 207.2 (C-19) and 50.6 (C-18). Compound 3 possessed a molecular formula of C 22 H 24 N 2 O 4 based on the HRESIMS. The 1 H and 13 C NMR spectra of 3 displayed similarity to 1 except for two methine signals at δ C 68.5 (C-19) and 47.3 (C-20) in 1 were substituted by double bond signals (δ C 136.1 and 119.8) in 3, suggesting that 3 was a dehydration product of 1.
Configuration of alkaloids 1-3 was determined by NMR and ROESY spectra together with their biogenetic pathway. The chemical shift of C-19 in 1 was deshielded (Δ2.6 ppm) similar to N 4 -demethyl-12-methoxyalstogustine and N 4demethylalstogustine, relative to 12-methoxyechitamidine and echitamidine due to intramolecular H-bonding between the nitrogen atom and the C-19-OH. 6a The ROESY correlations of H-3 with H-15 and H-20 in 1-3 placed them on the same sides. The double bond C-19/20 of 3 was determined as E according the ROESY correlation of H-19 with H-21. In the biogenetic pathway of akummicine, 10 the keto-enol tautomerism among O-C 17 -C 16 of preakuammicine would give two reaction routes, which led to alstrostines C-E and akummicine, respectively   , and two methoxyls (δ C 51.8, 51.5). Above data were similar to those of strictosidine 12 with exception for additional methylene (δ C 55.7, t) and methoxycarbonyl [δ C 172.9 (s) and 51.8 (q)] in 4. In the HMBC spectrum, both H-3 (δ H 4.07) and H-5 (δ H 3.08 and 3.12) were correlated with δ C 55.7 (t), suggesting the methylene was connected with N 4 . In addition, the methylene protons (δ H 3.47 and 3.60) showed correlations to 172.9 (s), indicating 4 was strictosidine methyl N 4 -acetate.
All alkaloids 1-17 were tested for their ability to prevent the cytopathic effects of cancer in breast cancer SK-BR-3, hepatocellular carcinoma SMMC-7721, human myeloid leukemia HL-60, pancreatic cancer PANC-1, and lung cancer A-549 cells, and their cytotoxicity was measured in parallel with the determination of antitumor activity using cisplatin as the positive control. Unfortunately, none of them showed positive activity (IC 50 > 40 μM).

Experimental Section
General Experimental Procedures. Optical rotations were measured with a Horiba SEAP-300 spectropolarimeter. UV spectra were recorded on a Shimadzu double-beam 210A spectrophotometer. IR (KBr) spectra were obtained on Bio-Rac. FTS-135 infrared spectrophotometer. 1 H, 13 C and 2D NMR spectra were recorded on a AM-400 and DRX-500 MHz NMR spectrometer with TMS as an internal standard. MS data were obtained on an API Qstar Pulsar I spectrometer. Silica gel (200-300 mesh) for column chromatography (CC) and GF 254 for TLC were obtained from Qingdao Marine Chemical Factory, Qingdao, China and sprayed with Dragdorff' reagent. C18 silica gel (20-45 μm) was bought from Fuji Chemical Ltd., Japan. MPLC was employed Buchi pumps system coupled with glass column (15 × 230 and 26 × 460 mm, respectively, C18 silica gel). HPLC was performed using Waters 600 pumps coupled with analytical and semipreparative sunfire C18 columns (150 × 4.6 and 150 × 10 mm, respectively). The HPLC system employed a Waters 2996 photodiode array detector and a Waters fraction collector II. Extraction and Isolation. Air-dried leaves and twigs (8.0 kg) of A. rostrata was crushed and extracted with EtOH (20 L × 3). After removal of the EtOH under reduced pressure, the residue was dissolved in 1% HCl, and partitioned with EtOAc for three times. The acidic solution was subsequently basified using ammonia water to pH 8~9, and partitioned with EtOAc for three times, affording a two-phase mixture including the aqueous phase, EtOAc/organic phase (total alkaloids). The

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-012-0019-y and is accessible for authorized users.