Cytotoxic sesquiterpene lactones from Vernonia pachyclada from the Madagascar rainforest.

In our continuing search for bioactive molecules from the rainforests of Madagascar as part of an International Cooperative Biodiversity Group (ICBG) program,2 an extract of the leaves of a Madagasacar collection showed moderate cytotoxicity against the A2780 human ovarian cancer cell line. This extract was selected for bioassay-guided fractionation based on its cytotoxicity and its presumed botanical novelty.3 The crude extract was purified by liquid-liquid partition, reversed-phase flash chromatography, and reversed-phased HPLC to yield three new sesquiterpene lactones designated glaucolides K-M. After this work was complete the plant was identified as Vernonia pachyclada Baker (Asteraceae). V. pachyclada is known locally as Tsaboraty, and is used for the treatment of wounds. Members of the Asteraceae family are well known for the production of sesquiterpenes such as the glaucolides, and their presence in the genus Vernonia is particularly well studied.4–9 The genus Vernonia has between 500 and 1,000 species, depending on taxonomic circumscription, with the majority of the species in tropical and warm parts of the Old World, but species are also found in North America. The genus is particularly rich in Madagascar, where nearly 100 species occur, the vast majority of which are endemic.10 Compound 1 was obtained as colorless crystals from methanol. HRFABMS indicated a molecular formula of C23H30O9. The 1H and 13C NMR data (Table 1), the HMQC correlations, and an HMBC experiment (Figure 1) indicated that compound 1 was a sesquiterpene with the same atom connectivity as that of the previously reported compound 8-O-desacetyl confertolide-8-O-methacrylate.11 However, the 1H NMR data for 8-O-desacetyl confertolide-8-O-methacrylate were not in agreement with those of compound 1 when both spectra were determined in the same solvent. Since no 13C NMR or optical rotation data were reported for the literature compound, these data could not be compared. The variations in the 1H NMR data indicated that the compounds are not identical, and suggested that the difference is a stereochemical one. Figure 1 Key HMBC correlations for 1 Table 1 1H and 13C NMR data for compounds 1–3 in CD3OD Therefore, the only remaining problem to solve was that of the stereochemistry of compound 1. The relative stereochemistry was determined based on an analysis of NOESY correlations (Figure 2). The C-15 protons and the C-6 and C-8 protons all correlated to each other, indicating that they have syn-orientations to each other. Also observed were NOESY correlations from the C-15, the C-14, and the C-8 protons to the C-1 proton. Surprisingly a NOESY correlation was also observed between the C-13 protons and the C-9 protons. This correlation was unexpected as the two positions appear to be some distance apart, but in some ring conformations the two positions do appear to be close enough to produce a correlation. Thus, the relative stereochemistry of compound 1 was determined. Figure 2 Key NOESY correlations for 1. During the course of the isolation, crystals of 1 were obtained from methanol. A crystal was thus selected and analyzed by single crystal X-ray diffraction. The results of this experiment confirmed the relative stereochemistry proposed based on the NOESY data and indicated an absolute stereochemistry of 1S,4R,5R,6S,8S,10S, as depicted (Figure 3). The relative stereochemistry for the known compound 8-O-desacetyl confertolide-8-O-methacrylate is 1S, 4R,5S,6S,8S,10R,11 so this compound and 1 differ in relative stereochemistry at C-5 and C-10. The structure was thus assigned as 1, and the compound was named glaucolide K. Figure 3 ORTEP diagram of 1. Compound 2 was obtained as a colorless amorphous solid. HRFABMS indicated a molecular formula of C21H28O8. The 1H and 13C NMR spectra clearly indicated that 2 was an analog of 1. The only significant differences in the 1H NMR data for 2 and 1 were the missing methyl signal at δH 1.99 and the shift of the methylene signal from δH 4.90 and 4.86 in 1 to δH 4.41 (d, J = 12.2 Hz) and 4.38 (d, J = 12.2 Hz) in 2. Thus it appeared as though compound 2 was the C-13 deacetate of 1. This hypothesis was supported by all of the spectral data and confirmed by acetylation of compound 2 to yield compound 1. The structure was thus assigned as 2, and was named glaucolide L. Compound 3 was obtained as a slightly yellow amorphous solid. HRFABMS indicated a molecular formula of C23H30O9. The 1H and 13C NMR spectra indicated that 3 was an analog of 1. No characteristic epoxide signals were observed in the 1H NMR spectrum of 3, and one additional signal was observed for a vinylic proton (δH 6.12, s). The 13C NMR spectrum also contained two additional signals for a C-C double bond. Thus it appeared as though compound 3 was the Δ5 4-hydroxy analog of 1. This hypothesis was supported by HMBC correlations (Figure 4) and the remaining spectral data. The configuration of the Δ5 double bond was assigned as E, as the only NOESY correlation observed from the C-5 proton was to the C-15 protons. No correlations were observed from the C-5 proton to the protons of the 8 or 9 positions. The remaining stereocenters were based on those found in 1. The structure was assigned as 3, and was named glaucolide M. Figure 4 Key HMBC correlations for 3. Compounds 1–3 were found to be cytotoxic in the A2780 human ovarian cancer cell line. Glaucolide M (3, 3.3 μM) was the most active compound, and was slightly more active than glaucolide K (1, 5.8 μM). Glaucolide L (2, 24.5 μM) was the least active.