Euglobal-IIIa, a novel acylphloroglucinol-sesquiterpene derivative from Eucalyptus robusta: absolute structure and cytotoxicity

Euglobal-IIIa (1), a novel acylphloroglucinol-sesquiterpene derivative, and a known analogue, have been isolated from leaves of Eucalyptus robusta. The structures was elucidated by extensive spectroscopic data and by comparison with data reported in literature, while the absolute configuration of 1 was determined by the X-ray diffraction analysis. Compound 1 exhibited comparable cytotoxicity with that of cisplatin against five human cancer cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW480 with IC50 values of 15.7, 15.5, 17.6, 14.3, and 21.8 µM, respectively. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-011-0021-9 and is accessible for authorized users.


Introduction
Euglobals are a group of caryophyllene-based monoterpenoid (or sesquiterpenoid) derivatives which have been found to be abundant in the genus Eucalyptus. 1 Pharmacological investigations of them showed antitumor, antimicrobial, and granulation inhibiting activities. 2 As part of our efforts to search for significant antitumor agents, a novel acylphloroglucinol-sesquiterpene derivative, named euglobal-IIIa (1), and a known analogure, were isolated from leaves of E. robusta Smith, a tree up to 20 meters distributed in Yunnan and Sichuan province, China. The structure of 1 was established on the basis of extensive spectroscopic methods and the absolute configuration was determined by the single crystal X-ray diffraction analysis, while the known compound was identified as sideroxylonal B (2) by comparison with data reported in literature. 3 The cytotoxicity of two compounds against five human cancer cell lines was evaluated.

Results and Discussion
An acetone extract of E. Robusta was partitioned between H 2 O and EtOAc. The isolation of the EtOAc lay afforded a new caryophyllene-based terpenoid, named as euglobal-IIIa (1), along with an analogue, sideroxylonal B (2).
Euglobal-IIIa (1), colorless crystals, was found to posssess a molecular formula of C 28 H 40 O 6 as assigned by HREIMS at m/z 472.2828 [M] + (calcd. 472.2825 [M] + ), implying nine degrees of unsaturation. The UV spectrum showed the existence of a phenyl group based on the maximum absorption bands at 282 and 232 nm, while the FT-IR spectrum exhibited absorption bands for carbonyl groups (1629 cm −1 ) and hydroxy groups (3556 and 3441 cm −1 ).
To establish the stereoconfiguration of 1, an ROESY spectrum was measured. In which, the cross peak between H-7 and H-14' suggested H-7 and Me-14' in the same side, and the cross peak of H-15' with H-6' suggested E form of double bond between C-4' and C-5'. However, the stereoconfiguration of C-1' and C-7' could not be determined according to the ROESY spectrum. Finally, an X-ray diffraction not only confirmed the structure of 1 but also established the absolute configuration of the whole molecule (Figure 1).
Both compounds 1 and 2 were evaluated for their cytotoxicity against five human cancer lines using the MTT method as reported previously. 5 Cisplatin was used as the positive control. The results showed that compound 1 displayed comparable cytotoxicity with that of cisplatin against SMMC-7721, A-549, MCF-7, and SW480, while compound 2 was inactive to all the tested strains (IC 50 > 40 μM) ( Table 2).

Experimental Section
General Experimental Procedures. Melting points were determined on an X-4 micro melting point apparatus. Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained by a Tenor 27 spectrophotometer with KBr pellets. 1D and 2D spectra were run on a Bruker DRX-500 spectrometer with TMS as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Mass spectra were recorded on a Waters AutoSpec Primier P776 instrument or an API QSTAR Pulsar i spectrometer. X-ray diffraction was performed on a Bruker SMART APEX-II diffractometer using graphitemonochromated Cu Kα radiation. Column chromatography (CC) was performed using silica gel (200-300 mesh and H, Qingdao Marine Chemical Co. Ltd., Qingdao, People's Republic of China). Fractions were monitored by TLC (GF254, Qingdao Marine Chemical Co. Ltd., Qingdao), and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH. All solvents were distilled prior to use.

Extraction and Isolation.
An air-dried sample (5 kg) was extracted in acetone at room temperature, and a crude extract was obtained after three times, which was partitioned between H 2 O and EtOAc. The EtOAc lay was separated by CC over silica gel (100-200 mesh, Qindao Marine Chemical Ltd., China) eluted with petroleum ether : acetone in a gradient of 0:1    Cytotoxicity Assay. Five human cancer cell lines, breast cancer MCF-7, hepatocellular carcinoma SMMC-7721, human myeloid leukemia HL-60, colon cancer SW480, and lung cancer A-549 cells, were used in the cytotoxic assay. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO 2 at 37 C. The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method in 96-well microplates. 5 Briefly, 100 µL adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with initial density of 1 × 10 5 cells/mL. Each tumor cell line was exposed to the test compound dissolved in DMSO at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM in triplicates for 48 h, with cisplatin (Sigma, USA) as a positive control. After compound treatment, cell viability was detected and a cell growth curve was graphed. IC 50 values were calculated by Reed and Muench's method. 6

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-011-0021-9 and is accessible for authorized users.