Kaurene Diterpenes and Other Chemical Constituents from Mikania stipulacea ( M . Vahl ) Willd .

O estudo fitoquímico de Mikania stipulacea conduziu ao isolamento e à identificação de lupeol, α-amirina, estigmasterol, β-sitosterol, campesterol, 3-Oβ-D-glicopiranosil estigmasterol, 3-OβD-glicopiranosil sitosterol, da cumarina isoescopoletina e dos ácidos vanílico, cinamoilgrandiflórico e ent-caurenóico, juntamente com dois diterpenos novos, os ácidos ent-9α-hidroxi-15β-E-cinamoiloxi16-cauren-19-óico e nt-9α-hidroxi-15β-Z-cinamoiloxi-16-cauren-19-óico. Estas substâncias foram identificadas com base na análise dos espectros de IV, EM e RMN de 1H e 13C.


Introduction
The tribe Eupatorieae forms the major part of the Asteraceae family and comprises nearly 2,400 species 1,2 .Mikania Willd is the only genus in the sub-tribe Mikaniinae of Eupatorieae.These species are native to Central and South America, where they are known as "guaco" and used to treat fever, rheumatism, flu and respiratory tract diseases 3,4 .As might be expected for such a large and diverse genus, its chemistry is also diverse 5 .Less than 10% of the more than 430 known Mikania species have been studied.Sesquiterpene lactones are found in nearly two thirds of these species 5 .In the other species whose chemistry has been investigated, diterpenes, mainly of the ent-kaurene type, predominate 6 .
Continuing our work on this genus 7,8 we report herein the isolation of two new kaurene-type diterpenes from M. stipulacea.Their structures were proposed on the basis of spectroscopic data and comparisons of the attributed signals with previously published data on related compounds.

General
The IR spectra were obtained on KBr pellets in a Perkin Elmer model 1420 spectrophotometer. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were recorded on a Bruker DPX 300 in CDCl 3 with TMS as internal standard.EIMS was obtained at 70 eV on HP 5988-A.Optical rotations were measured with a Polartronic HH8.Prep.TLC was carried out on Si gel PF-254 (Merck), CC on Si gel 60 (0.063 a 0.200) (Merck) and VLC on Si gel 60 H (0.005 -0.045) (Merck).

Plant material
Mikania stipulacea (M.Vahl) Willd was collected in Restinga de Maricá, Rio de Janeiro, RJ, Brazil, in July, 1996, and identified by Professor Dr. Janie G. Silva (Instituto de Biologia da Universidade Federal Fluminense -Rio de Janeiro).A voucher specimen (SPFR 04123) was deposited in the herbarium of the Department of Biology, FFCLRP/ USP and was used for the authentication of the species.

Extraction and fractionation
Dried and powdered whole M. stipulacea plants (1.7 kg) were exhaustively extracted at room temperature with hexane, ethyl acetate and ethanol.Evaporation of the solvents under reduced pressure furnished 46.0 g, 23.0 g and 225.0 g respectively, of crude extracts.The bulk of each extract (45.0 g hexane, 22.0 g ethyl acetate, 60.0 g ethanol) was chromatographed separately over silica gel under vacuum (VLC) and eluted with hexane, gradually increasing the polarity with ethyl acetate and then methanol.
From the crude hexane extract, 14 fractions were collected after VLC.
Finally, fraction 10 (500.0 mg) after CC on silica gel (hexane-ethyl acetate and methanol in mixtures of increasing polarity) and precipitation in ethyl acetate of subfr.10.6 led to the isolation of a mixture of β-sitosteryl glucopyranoside and stigmasteryl glucopyranoside (4.0 mg).
From the crude ethyl acetate extract, 15 fractions were collected after VLC.
From the crude ethanol extract, 15 fractions were collected after VLC.
The ent-kaurene diterpene 1, obtained as a pure solid exhibited a 1 HNMR spectrum with typical signals of the ester E-cinnamate, δ 7.69 d and 6.46d (AB system, J AB 16 Hz).The H-15 exhibited an even higher paramagnetic shift when compared with the H-15 chemical shift of entkaurenoic acid (3) (δ 2.09) and of cinnamoylgrandifloric acid (4) (δ 5.41), (Table 1).The stereochemistry at C-9 and C-15 was deduced from the chemical shift of H-15 which could be explained only by a deshielding effect of a β-oriented oxygen function in C-9.  2. 13 C NMR data (δ) for 1 and 2 in CDCl 3 at 75 MHz compared with 4 (CDCl 3 ) 9 , 5 (C 5 D 5 N) 17 and 6 (C 5 D 5 N) 18 .28.9 q 028.9 q 28.9 q 029.6 q 29.3 q 19 183.0 s0 A comparison of the 13 C NMR spectral data of 1 and 4 showed that the hydroxyl group at C-9 in 1 produced a deshielding effect on the signals of C-8, C-10 and C-11 and a shielding effect on the signals of C-1, C-5, C-7 and C-12 (γgauche effect), thus confirming the position and the stereochemistry proposed earlier for C-9 (Table 2).In accordance with this conclusion, the 13 C NMR data of 1 were similar to those for ent-9α-hydroxy-16-kauren-19-oic acid (5).Some differences were due to the additional O-cinnamoyl group at C-15, which caused a strong high frequency shift of this carbon.In addition, direct comparison of 1 with 4 and grandifloric acid (6), in which the C-9 (without the hydroxyl group) appears respectively at δ 53.1 and 54.0, confirmed the kaurene skeleton with an O-cinnamoyl group at C-15 and an OH group at C-9.
The axial trans position of the C-19 carboxyl group in relation to H-5 was deduced from the chemicals shifts of C-5, C-18, C-19 and C-20 16 .
The ent series of compound 1 was established by measurement of the optical rotation.The structure was confirmed by MS and IR data.
The diterpene 2 was obtained mixed with compound 1.The presence of the E and Z-cinnamate esters in the diterpenes was suggested by the observation of their typical signals, respectively, at δ 7.69 d and 6.46d (AB system, J AB 16 Hz) and δ 6.96 d and 5.96 d (AB system, J AB 12.8 Hz) in their 1 HNMR spectrum.1 and 2 were 2',3' -E/Zisomers which could be identified by their different chemical shifts and couplings.
de São Paulo (FAPESP) for financial aid and a fellowship to A.M.N.
FAPESP helped in meeting the publication costs of this article.On the basis of the present data, we conclude that M. stipulacea belongs to a fairly large group of Brazilian Mikania species which produce ent-kauranes rather than sesquiterpene lactones 5,7 .