Eudesmane Derivatives from Verbesina turbacensis

Species of the genus Verbesina (Asteraceae, tribe Heliantheae, subtribe Ecliptinae) have produced a range of eudesmane sesquiterpenes with cinnamate or a derived ester group, of which, αand β-verbesinolcoumarates were the first reported examples. Several species also afforded elemanolides, diterpenes, flavonoids, and biological active guanidines as Galegine, the toxic principle of V. enceloides Benth. As a continuation of our phytochemical investigation on Venezuelan Compositae, in this paper we describe the results of our study of Verbesina turbacensis H.B.K., a previously uninvestigated species, which is widely distributed throughout of Central America from Mexico to Colombia and Venezuela. V. turbacensis as well as V. caracasana Fries, a plant which contains the dosedependent hypotensive agent Caracasanamide, are frequently used in Venezuela as medicinal plants against a variety of diseases.


Introduction
Species of the genus Verbesina (Asteraceae, tribe Heliantheae, subtribe Ecliptinae) have produced a range of eudesmane sesquiterpenes with cinnamate or a derived ester group, [1][2][3][4][5][6][7] of which, αand β-verbesinolcoumarates were the first reported examples. 1,8Several species also afforded elemanolides, 9,10 diterpenes, 11 flavonoids, 12 and biological active guanidines as Galegine, the toxic principle of V. enceloides Benth. 13,14s a continuation of our phytochemical investigation on Venezuelan Compositae, [15][16][17] in this paper we describe the results of our study of Verbesina turbacensis H.B.K., a previously uninvestigated species, which is widely distributed throughout of Central America from Mexico to Colombia and Venezuela. 18V. turbacensis as well as V. caracasana Fries, a plant which contains the dosedependent hypotensive agent Caracasanamide, 19 are frequently used in Venezuela as medicinal plants against a variety of diseases.

Results and Discussion
The dried, ground leaves and stems of V. turbacensis were extracted with acetone at room temperature and the obtained extract was purified by standard procedures.Vacuum liquid chromatography separation over silica gel 20 of the fractions obtained from a hexane-EtOAc (3:1, v/v) eluate, gave the sesquiterpenes 1, 3, 5 and 9.
Compound 1 was crystallized from acetone as colourless prisms (mp 221-223º C; [α] D : + 147.2º) and analysed for C 24 H 32 O 4 ([M + ] m/z 384.2189 high resolution EI-MS).Its 1 H NMR spectral data were in close agreement with those reported by Bohlmann and Lonitz 2 for the methyl-6β-[cinnamoyloxy]eudesman-15-oate 2. However the IR, 1 H NMR and 13 C NMR spectra of compound 1 revealed the presence of a free carboxyl group [IR: 3600-3200 and 1735 cm -1 ; δ H 11.45 broad singlet; δ C 181.10 (O=C-O-)], instead of the methyl ester present in 2. In accordance with above assignments, treatment of 1 with CH 2 N 2 /eter gave 2 and consequently 1 was identified as 6 β-[cinnamoyloxy]-eudesman-15-oic acid.Further support to structure 1 was provided by 1 H, 1 H-COSY, HMQC and HMBC experiments, which allowed the assignments in Tables 1 and  2. The NOESY spectrum of 1 showed cross peaks between the signal assigned to H-6 and the signals of H-4 and H-7 respectively, suggesting that all three protons were on the same side of the molecule.Assuming an A/B ring fusion (5αH, 10βMe); these results clearly confirm a β-configuration for C-4 carboxyl-, C-6 "trans"-cinnamoyl-and C-7 isopropyl groups.
Compound 5 was isolated as colourless hexagonal plates (mp185-87º C; [α] D : +65.89º).On the basis of high resolution EI-MS (m/z 368.2375 [M-H 2 O] + ) and 13 C-NMR (BB and DEPT) spectral data, the molecular formula of 5 was deduced to be C 24 H 34 O 4 .These data, as well as 2D-NMR experiments, revealed that 5 is a 6β-[cinnamoyloxy]eudesmane sesquiterpene similar to 1, with an additional secondary hydroxyl group (δ H 3.10 (-OH) and 3.44 (H-C-O-); δ C 78.84), but in which, the C-15 carboxyl group was replaced by a methyl (δ H 1.14; δ C 17.92) located on a carbon that supports a tertiary hydroxyl group (δ C 75.18).The position of secondary hydroxyl group was clarified by the correlation peaks between C-1/H-3; C-2/H-3; C-4/ H-3; C-5/H-3; C-15/H-3; C-3/H-1; C-3/H-2; C-3/H-5 and C-3/H-15 in the HMBC spectrum.On acetylation with Ac 2 O/Py, compound 5 gave the monoacetate 6. Treatment of 5 with H 2 SO 4 in dry acetone gave the dehydration products 7 and 8, but did not give an acetonide derivative.In spite of this result, the stereochemistry in C-3 was evidenced on the basis of H-3 multiplicity signal and the magnitude of its coupling constants to H-2α (J 3.5 Hz) and H-2β a (J 11.5 Hz), which were in agreement with a βequatorial oriented hydroxyl group.All the above data were consistent with the structure proposed for 5, and these ones also agree well with the spectroscopic data reported for the 6β-[cinnamoyloxy]-3β, 4α-dihydroxyeudesmane, a sesquiterpene previously isolated from V. persicifolia D.C. 6 and V. oncophora Rob.et Seat. 7ompound 9 (oil, [α] D : -13.8º, formula molecular C 24 H 34 O 4 ) was also identified as a 6β-[cinnamoyloxy]eudesmane derivative similar to 5. The 1 H-and 13 C-NMR data, as well as the carbon connectivity pattern deduced from 1  B and H-3α revealed a β-axial orientation of hydoxymethyl group.The physical constants and the EIMS and 1 H-NMR data of 9, were in agreement with those published for the 6β-[cinnamoyloxy]-4α, 15-dihydroxyeudesmane, which has been previously isolated from V. eggersii. 5Until now 13 C-NMR data of 9 have not been reported in the literature.2][23] From a biological point of view, it is also important to highlight that many eudesmane derivatives, with structures related to 1-9, have shown antifeedant, 24 antibacterial 25 or cytotoxic 26 properties.

General experimental procedures
Melting points were determined with a Fisher-Johns apparatus and they have not been corrected.Optical activities were measured in CHCl 3 on a Rudolph Research Autopol III polarimeter.IR spectra were taken on a Perkin-Elmer FT-1725X spectrophotometer as film or KBr pellets. 1 H, 13 C and two-dimensional NMR spectra were measured on a Bruker-Avance DRX400 instrument, using CDCl 3 as solvent with TMS as internal standard.EI-MS and HREI-MS were run on a Hewlett-Packard 5930A and on an Autospec VG spectrometer, respectively; direct inlet, 70 eV.TLC was carried out on 0.25 mm layers of silica gel PF 254 (Merck).VCC was performated with silica gel 60 (70-230 mesh.).

Extraction and isolation of the constituents
Leaves and stems of V. turbacensis (ca 7.5 kg) were airdried, ground and exhaustively extracted with acetone at room temperature for 1 week.The dissolution obtained was concentrated in vacuo to afford a dark brown residue (450 g), which was preadsorbed on silica gel and subjected to VCC over silica gel, 20 using hexane with increasing amounts of EtOAc as eluent.Fractions of 1 L were collected and combined based upon TLC monitoring.From fractions eluted with hexane-EtOAc (7:3, v/v), stigmasterol (55 mg) was purified by crystallisation and identified by mp, IR, 1 H NMR and TLC comparison.Fractions eluted with hexane-EtOAc (3:2, v/v) were purified by repeated flash chromatography or preparative TLC yielding four compounds, which were obtained in the following sequence: 1 (2.83 g), 3 (870 mg), 5 (6.25 g) and 9 (35 mg).
H, 1 H-COSY, HMQC and HMBC experiments, indicated that 9 lacks of the C-3 hydroxyl group and that the C-4 methyl has been replaced by a hydroxymethyl group [δ H 3.35 d (J 11.0 Hz) and 3.75 dd (J 11.0 and J 1.3 Hz); δ C 62.73].The W coupling (J = 1.3 Hz) between H-15