A New Cytotoxic 19-Nor-cardenolide from the Latex of Antiaris toxicaria

A new nor-cardenolide, named toxicarioside H (1), was isolated from the latex of Antiaris toxicaria (Pers.) Lesch (Moraceae). Its structure was elucidated on the basis of HRFAB-MS and spectroscopic techniques (IR, UV, 1D and 2D NMR). Compound 1 showed significant cytotoxicity against K562, SGC-7901, SMMC-7721, and HeLa cell lines in vitro by MTT method.


Introduction
The latex of Antiaris toxicaria (Pers.) Lesch (Moraceae) has been the main source of the poisonous principle in dart and arrow poisons prepared throughout southeastern Asia, from Burma to China and Indonesia [1]. Its toxicity is due to the presence of a complex mixture of cardenolide glycosides [2,3]. Previous studies of the toxicity of this plant in Indonesia or Malaysia led to the isolation of cardenolides from the latex, seeds, and stems [4,5]. In our previous work on screening for cytotoxic OPEN ACCESS agents from tropical medicinal plants in Hainan Province, China, three new cytotoxic cardenolides named toxicarioside E, F, and G have been isolated from the latex of A. toxicaria [6,7]. In continuation of our search for cytotoxic constituents, a new nor-cardenolide, named toxicarioside H (1) was obtained. Compound 1 showed potent growth-inhibitory activity on K562, SGC-7901, SMMC-7721, and HeLa cell lines. The present paper discusses the structural elucidation and cytotoxicity of this new compound.

Results and Discussion
Compound 1 was obtained as a white amorphous powder. (d, 1H, J = 7.9 Hz, H1') in the 1 H-NMR spectrum indicated that 1 was a glycoside incorporating a sugar unit with -linkage. Comparing the 1D-NMR spectral data with those of the toxicarioside A showed that the aglycone of 1 had no aldehyde group [8], while C-10 ( 75.2) was substituted by a hydroxyl group. This was further confirmed by the correlations from H2 (1.84, 1.64) and H4 (2.02, 1.38) to C10 ( 75.2) in the HMBC spectrum. Thus, the aglycone of 1 was determined as 10-hydroxy-19-nor-antiarigenin.
The sugar unit of 1 was identified mainly by analysis of 1 H-NMR and 1 H-1 H COSY spectra. 1 H-1 H COSY spectrum of 1 allowed unambiguous assignment of the entire H1'-H6' spin system, the relative configuration of C1'-C5' centers could be deduced by analysis vicinal coupling constants for the sugar proton signals. Thus, a H1'/H2' diaxial relationship was indicated by the 7.9 Hz coupling constant observed at H1', while the small, second coupling constant observed in the well resolved double doublet signal for H2' ( 3.01, J = 7.9, 2.8 Hz) demonstrated that H3' was equatorial. Likewise, the small coupling constant observed in the triplet signal for H3' ( 4.24, J = 2.7 Hz) demonstrated that H4' was axial, and the large coupling constant observed in the double doublet signal for H4' ( 3.14, J = 9.6, 2.8 Hz) demonstrated that H5' was also axial. In the HMBC spectrum, the correlation from H-OCH 3 ( 3.41) to C2' ( 81.8) (Figure 2) suggested that C2' was the site for attachment of the O-methyl ether. Hence, the sugar moiety was determined as a rare sugar javose. The HMBC correlations between H3 ( 4.11) and C1' ( 98.1) (Figure 2) suggested that the sugar moiety was linked to C3. The relative stereochemistry of 1 was determined by ROESY correlations (Figure 2). The sugar moiety of 1 was determined as D-javose by acid hydrolysis. Based on the above evidence, compound 1 was identified as 10-hydroxy-19-nor-antiarigenin-3-O--D-javopyranoside, named toxicarioside H.  In addition to the effect of the cardenolides on the activity of the ubiquitous cell surface enzyme Na + /K + -ATPase, recent studies have demonstrated that this kind of compounds could inhibit the growth of cancer cells. We have, therefore, tested the isolated compounds for their cytotoxicity. The cytotoxicity of compound 1 was evaluated in vitro using the MTT method [9]. Compound 1 showed significant cytotoxicity against the K562, SGC-7901, SMMC-7721, and HeLa cell lines with the IC 50 values of 0.0040.037 μg/mL, and mitomycin C was used as a positive control.

General
Melting points were obtained on a Beijing Taike X-5 stage apparatus and are uncorrected. Optical rotation was recorded using a Rudolph Autopol III polarimeter (Rodolph Research Analytical, New Jersey, USA). The UV spectra were measured on a Shimadzu UV-2550 spectrometer. The IR spectra were obtained on a Nicolet 380 FT-IR instrument, as KBr pellets. The NMR spectra were recorded on a Bruker AV-400 spectrometer, using TMS as an internal standard. The HRFABMS spectra were measured with a VG Auto-Spec-3000 mass spectrometer. Column chromatography was performed with silica gel (Marine Chemical Industry Factory, Qingdao, P.R. China). TLC was preformed with silica gel GF254 (Marine Chemical Industry Factory, Qingdao, P.R. China).

Extraction and isolation
Four L of latex of A. toxicaria was extracted three times with 95% EtOH (eight L) at room temperature and then filtered. The combined extract was evaporated in vacuo to yield a syrup (263.8 g), which was suspended in H 2 O and partitioned successively with petroleum ether and EtOAc to afford a petroleum ether extract and EtOAc extract. The EtOAc extract (8.68 g) was chromatographed over a silica gel column by gradient elution utilizing CHCl 3 and MeOH as solvent system to give sixteen fractions. After repeated silica gel column chromatography (CHCl 3 -MeOH 14:1), fraction 15 (1.24 g) afforded compound 1 (11.3 mg).

Cell cultures and in vitro cytotoxicity assay
The human myeloid leukemia cell line (K562), human gastric cell line (SGC-7901), human hepatoma (SMMC-7721), and human cervical cancer (HeLa) cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology. Cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin sulfate at 37 ºC, 5% CO 2 . The MTT assay was performed according to the method described in previous literature [9]. The IC 50 values are listed in Table 2.