A New Megastigmane Diglycoside from Litsea glutinosa ( Lour . )

Estudo fotoquímico das folhas e galhos de Litsea glutinosa (Lour.) C. B. Rob. forneceu um novo diglucosídeo megastimano (6S, 7E, 9R)-6, 9-dihidroxi-4, 7-megastigmadieno-3-ona9-O-[a-L-arabinofuranosil-(l→6)]-b-d-glucopiranosídeo (1), juntamente com os glicosídeos (6S, 7E, 9R)-roseosido (2), (7’R, 8’R)-3, 5’-dimetoxi-9, 9’-dihidroxi-4, 7’-epóxi lignina 4’-b-D-glucopiranosídeo (3), álcool (7’R, 8’S)-dihidrodehidrodiconifenil 9’-O-b-Dxilopiranosídeo (4) e pinoresinol 3-O-b-D-glucopiranosídeo (5). Suas estruturas foram estabelecidas com base em métodos espectroscópicos e químicos. Compostos 2-5 são reportados pela primeira vez nestas espécies. Composto 1 foi avaliado para atividades citotóxicas de linhagens de células tumorais humanas (células leucemia mielóide HL-60, carcinoma hepatocelular SMMC-7721, câncer de pulmãoA-549, câncer da mama MCF-7 e câncer do colo SW480), para as quais provou-se ser inativo (IC50 > 40 mM).


General
Commercial silica-gel plates (Qing Dao Marine Chemical Group Co.) were used for thin layer chromatography (TLC) analyses.Normal silica was used for normal (Qing Dao Marine Chemical Group Co.) and reversed-phase CC (Merk).UV spectra were measured on a Shimadzu UV-2401PC spectrophotometer (l max in nm).Optical rotation was obtained on a Horiba SEAP-300 spectropolarimeter.Infrared (IR) spectra were measured on a Bio-Rad FTS-135 infrared spectrophotometer (ν max in cm -1 ). 1 H (500 MHz) and 13 C NMR (125 MHz) spectra, as well as 2D-NMR spectra, were recorded on a Brucker DRX-500 spectrometer, chemical shifts d in ppm related to TMS (coupling constant J in Hz).Electrospray ionization mass spectrometry (ESI-MS) spectra were acquired on a VG-Autospec 3000 mass spectrometers.

Acid hydrolysis of compound 1
A solution of 1 (1 mg) in 1 mol L -1 HCl (2 mL) was heated at 80 o C for 2 h.The reaction mixture was diluted with H 2 O and then extracted with Et 2 O. D-glucose and L-arabinose were identified from the aqueous phase by TLC comparison with authentic substances.

Bioassays
Human cancer cell lines myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549, breast cancer MCF-7 and colon cancer SW480 cells were used in the cytotoxic assay, which was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-3,5-di-phenytetrazoliumromide) method in 96-well microplates. 16Cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA) and supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO 2 at 37 o C. The 1 H NMR spectrum of 1 showed signals assignable to a double bond proton at d 5.90 (s), two vinyl protons at d 5.85 (d, J 15.7 Hz) and d 5.84 (dd, J 15.7 and 6.4 Hz), an oxygenated methine at d 4.42, an isolated methylene (d 2.54 and 2.17, each d, J 17.0 Hz), a vinyl methyl (d 1.92, d, J 1.5 Hz), a secondary methyl (d 1.29, d, J 6.4 Hz) and two tertiary methyl singlets at d 1.04 and 1.03 (Table 1).The 13 C NMR spectrum of 1 showed 24 carbon signals, including those characteristic of a disaccharide moiety.Analysis of 1D and 2D NMR spectra indicated that the aglycone portion of 1 was identical to that of the megastigmane glycosides (6S, 7E, 9R)-roseoside (2). 17 1H and 13 C NMR data of the disaccharide moiety suggested the presence of glucopyranosyl and arabinofuranosyl units. 17,18This was confirmed by acid hydrolysis of 1 that afforded D-glucose and L-arabinose, which were identified by direct comparison with authentic samples.The J values of the anomeric protons at 4. 35 (d,  The locations of the sugar residues in 1 were established by the presence of the NOESY (nuclear Overhauser enhancement spectroscopy) cross peak between H-1' (d 4.35) and H-9 (d 4.42), and HMBC (heteronuclear multiple bond correlation) correlation between H-1' and C-9 (d 76.8).These results demonstrated the glucosyl moiety are connected to the 9-OH group of the aglycone through a glycosidic linkage. 17Furthermore, the C-6' signal of the inner b-D-glucopyranosyl unit was observed at d C 68.0, indicating the involvement of this carbon in a glycosidic linkage. 19The other anomeric proton (arabinose) at d 4.94 was also correlated with C-6' of glucose at d 68.0 (Figure 2).From the above data, compound 1 appears to be the arabinofuranosyl-(l"→6')-glucopyranosyl analogue of roseoside.Literature data report that most megastigmane glycosides possesses the 1"→6' linkage in their disaccharide unit. 19,20tereochemical assignments of 1 could be deduced from NOESY, COSY and CD (circular dichroism) experiments.The 1 H-1 H COSY spectra showed longrange couplings between H-2b and H 3 -11, H-2a and H-4, whereas NOE (nuclear Overhauser effect) cross-peaks  2).These results demonstrated that the side-chain (C-7-C-10) was in a pseudo-axial orientation to the half-chair conformation of the cyclohexenone ring.The CD spectrum of 1 showed a positive Cotton effect at 240 nm, similar to that of 2, thus indicating a 6S-configuration.The absolute configuration at C-9 of the aglycon was assigned as R on the basis of a diagnostic chemical shift of the C-9 signal (d 76.8) in the 13 C NMR spectrum. 21Consequently, the structure of 1 was determined to be (6S, 7E, 9R)-6,9-dihydroxy-4,7-megastigmadien-3-one-9-O-[a-L-arabinofuranosyl-(l"→6')]-b-d-glucopyranoside (Figure 1).Earlier study reported the isolation of (6S, 7E, 9R)-6, 9-dihydroxy-4, 7-megastigmadien-3-one-9-O-[a-L-arabinopyranosyl-(l→6)]-b-d-glucopyranoside from Lonicera gracilipes Var.glandulosa. 22e structures of known compounds 2-5 were identified as (6S, 7E, 9R)-roseoside (2), 17 5), 24 by comparison of physical data and spectroscopic literature data.These glycosides are reported for the first time from the genus Litsea.

Results and Discussion
Naturally occurring megastigmane derivatives are an expanding class of compounds.6][27][28] Compound 1 was tested for its cytoxicity effects in the human tumor cell lines myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549, breast cancer MCF-7 and colon cancer SW480 cells, but it proved to be inactive (IC 50 > 40 mM).

Compound 1 ,
[a] D 21 -16° (c 0.55, CH 3 OH) was isolated as an amorphous powder.The molecular formula C 24 H 38 O 12 was deduced from the high resolution fast atom bombardment mass spectrometry (HR-FABMS) exhibiting a quasi-molecular ion at m/z 517.5513 [M-H] -(calcd.for 517.5525).Compound 1 showed UV absorption at 250 nm and an IR band at 1642 cm -1 , characteristic of an a,b-unsaturated carbonyl.