Norsesquiterpenoids from the leaves of Croton tiglium

Two new compounds, badounoids A (1) and B (2), together with 13 known norsesquiterpenes, were isolated from the leaves of Croton tiglium L. The structures of the new compounds were established by means of spectroscopic methods. The absolute configuration of badounoid B was determined by single-crystal X-ray diffraction analysis. All the known compounds were isolated from Croton plants for the first time which added a new chemical facet for this genus. The selected compounds were evaluated for their cytostatic activity against several cancer cell lines. None of them was found to be active. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-011-0035-3 and is accessible for authorized users.


Introduction
The intriguing structures of Euphorbiaceae plants and their diverse biological activities have attracted great interest in the recent years. 1 Croton tiglium L. is a plant belonging to the family Euphorbiaceae, its seeds, a well-known traditional Chinese medicine have been extensively investigated. So far, diterpenoids, alkaloids, flavonoids, and steroids have been characterized from the seeds, they were found to have antitumor, antiinflammatory, analgesic, and lipid lowering effects. 2 The leaves of C. tiglium have been used to treat diarrhea, tinea, pain, and hurt, 3 however, little is known for its chemical profiling. During our search for active compounds from the leaves, fifteen norsesquiterpenes including two new ones were isolated and structurally identified. This paper describes their isolation and structural identification.

Results and Discussion
Badounoid A (1), isolated as colorless gums, had the molecular formula C 14 H 20 O 3 derived from its positive HRESIMS at m/z 259.1318 [M + Na] + (calcd. 259.1310), indicating five degrees of unsaturation. The IR spectrum showed the absorption bands for hydroxy (3431 cm −1 ) and ,-unsaturated carbonyl (1654 cm −1 ) groups. The 13 C NMR and DEPT spectra revealed 14 carbon resonances, which are four methyl, one oxygenated methylene, four methine, and five quaternary carbons (including one oxygenated carbon and one carbonyl), indicating that 1 is an analogue of 5. The 1 H-1 H COSY correlation of H-2 ( 6.99)/H-3 ( 6.18), and HMBC correlations of H-2, H-3, Me-14/C-4 ( 188.9), Me-14/C-5 ( 131.3), C-6 ( 161.9), and Me-12/C-1 ( 41.0), C-6 ( Figure 1) suggested the west part of 1 as shown. The side chain of 1 was identified as a substituted isoprenyl group according to the following evidence: (i) 1  The configuration at C-9 still remained unresolved, since the stereochemistry determination at the chiral center of the conformationally flexible chain is always challengeable. Thus, the structure of 1 was deduced as shown, with a trivial name badounoid A.
Badounoid B (2) was isolated as colorless crystals. The molecular formula of 2 was determined to be C 13 H 24 O 3 from its HRESIMS at m/z 227.1652 [M  H]  (calcd. 227.1647), requiring two degrees of unsaturation. The IR spectrum displayed the existence of OH (3430 cm −1 ) and C=C (1638 cm −1 ) functionalities. The NMR data of 2 resembled those of 6. Interpretation of 1 H-1 H COSY, HSQC and HMBC spectra of 2 disclosed that compounds 2 and 6 have the same planar structure. The ROESY correlation of H-5/H-7 suggested that Me-13 and OH-6 were spacially vicinal. Whereas, the scarcity of diagnostic ROESY signals made it difficult to assign the relative configuration at C-3. Thus, the configurations at C-3 and C-9 of flexible side chain were clarified by X-ray diffraction using Cu-irradiation ( Figure 2), which also allowed the assignment of absolute configuration in 2 as 3R, 5R, 6S, and 9R. Therefore, the structure of 2 was determined as shown and given a name badounoid B.
Megastigmane norsesquiterpenoids have been widely found in the plants. However, their real role in the plants or in drug discovery is poorly known. It was reported that this type of norsesquiterpene possesses antiinflammatory activity. Whether the present isolates being also responsible for the traditional uses of the leaves in infectious diseases needs further investigation. In this study, the selected compounds (1, 3, 4, 1214) were evaluated for their cytostatic activity against HL-60, SMMC-7721, A-549, MCF-7, and SW480 human cancer cells, however, all these compounds showed no activity in this assay.
Among these miscellaneous compounds, we noted that the position of OH may be at C-3, C-4, C-5, C-6, or C-9.The OH group at C-3 is readily oxidized into a ketone when a double bond is formed between C-4 and C-5. Likewise, the OH-3 tends to be eliminated when a ketone occurs at C-4. In this sense, we could tentatively conclude that compounds 5, 8, 9, and 13 are probably unstable when they are exposed at oxidative environment.
Cytostatic Assay. The cytostatic assay was performed using the MTT method, as previous method with slight modification. 15,16 Briefly, human tumor cells were seeded into 96-well plates and permitted to adhere for 12 h before drug addition. For suspended cells, they were seeded immediately before drug addition with an initial density of 12  10 5 cells/mL. Each cell line was incubated with different concentrations of the compounds for 48 h. DDP and taxol were used as positive controls. Cell viability was measured and IC 50 values were calculated.

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-011-0035-3 and is accessible for authorized users.