The Use of 13 C and 1 H-NMR in the Structural Elucidation of a New Nor -Lupane Triterpene

Tomando-se por base as evidências espectroscópicas, RMN 1 H, 13 C, experimentos de RMN 2D, em especial correlações homonuclear 1 H- 1 H, heteronuclear 1 H- 13 C e correlações a várias ligações (HMBC), sua estrutura foi elucidada como: 29-hidroxiplatan-28-ato de β -D-glicosila. The new nor -lupane triterpene, isolated from the leaves of Eugenia florida DC (Myrtaceae) was identified on the basis of its spectroscopic data, 13 C and 1 H-NMR-2D, specially COSY 1 H- 1 H, COSY 1 H- 13 C and HMBC correlations, as 28-O- β -D-glucopyranosyl ester of 29-hydroxyplatanic acid.


Introduction
The triterpenes are a large group of plant substances with a wide spectrum of biological activities, such as anti-HIV 1 . The study of the Eugenia florida DC (Myrtaceae) leaves, which occur in the areas of tropical and subtropical 2 climate, is underway in our laboratories. In this work an unpublished triterpene with skeleton nor-lupane was identified. The identification of its structure was made through the use of spectroscopic methods. The homonuclear 1 H-1 H and one bond and multiple bond heteronuclear 1 H-13 C correlations experiments were used as the main tools to identify this new compound.

Results and Discussions
The structural determination of 1 was done on the basis of a comparative analysis of its 1 H and 13 C-NMR 1D and 2D data with those reported in the literature for platanic acid 1,3,4 . In the 1 H-NMR spectrum of 1, it was observed the presence of 5 methyl signals, δ 0.77, 0.85, 0.94, 0.96 and 1.07 (Table 1). The signal at δ 2.21 of the CH 3 -29, which is characteristic of platanic acid 1 was not detected. At δ 3.26, it was observed a triplet (J = 7.6 Hz) of the H ax bounded to C-3, which was coupled with H-2. The multi-plet appearing at δ 3.36-3.43 corresponds to H-19, coupled with H-18 and H-21. The COSY 1 H-1 H spectrum of 1 ( Fig.  1) also exhibited several important correlations to establish the triterpene skeleton and the attribution of the 1 H chemical shifts (Table 1) was supported by these data. From the DEPT 135 experiment it was possible to determine that 1 was a primary alcohol, which justifies the fact that 1 showed only 5 methyl groups, instead of 6 as in platanic acid. The location of the hydroxyl group at CH 2 -29 (δ 68.6), whose 1 H signals appeared as two doublets at δ 4.40 and 4.32 (J = 18.8 Hz) and the attribution of the chemical shifts of all methyl groups, was carried out by analysis of the HMBC spectrum (Figs. 2 and 3). It was noticed the unequivocal correlation of the two methyl groups, H-23 (δ 1.07) and H-24 (δ 0.85) with C-3 at δ 78.1 ( 3 J, Table 1), and with the carbons C-5 at δ 55.7, and C-4 at δ 39.2 ( 2 J). It was also noticed the correlation of the methyl H-25 (δ 0.77) with C-10 at δ 37.4 ( 2 J) and of two methyl groups, H-26 and H-27 with C-8 at δ 40.9 (respectively 2 J and 3 J). H-29 showed correlation with the carbonyl at δ 214.2 (C-20) 3 .
The presence of the sugar moiety in the molecule was verified by the signal of the anomeric hydrogen detected at Article δ 5.95 (d, J = 8.4 Hz), confirmed by other signals between δ 3.70 and 4.36. The inspection of the COSY 1 H-1 H spectrum ( Fig. 1), relatively to the glucosyl unit, allowed to establish the correlation between the hydrogens H-1' to H-6'. The inspection of the HMQC spectrum of 1 (Fig. 2, Table 1) showed correlation of H-1'-H-6' signals with the signals at δ 95.5, 73.6, 78.3, 70.9, 78.2 and 62.0, C-1' to C-6' respectively. These signals and these correlations are the same registered in the literature for β-D-glucose 7 . After having assigned the 1 H and 13 C chemical shifts of the sugar moiety, it was still remaining to locate this unit in the nor-lupane skeleton at the position C-3, C-28 or C-29. The 13 C-NMR spectrum showed a carbonyl carbon signal at δ 174.7, shielded by 4.0 ppm, which occurs when C-28 is an ester function 5,6 . This functionality was confirmed by the HMBC spectrum (Table 1, Fig. 3), whose 1 H signal at δ 5.95 was correlated to the carbonyl carbon at δ 174.7 ( 3 J). This unequivocally confirmed that the position C-28 was indeed esterified with β-D-glucose.
Finally, the mass spectrum of 1, obtained by FAB/MS, revealed the peak corresponding to the molecular ion (m/z 637, M+1, 3%), and the peaks 636 (5) These data allowed to attribute the structure of 1 as being 28-O-β-D-glucopyranosyl ester of 29-hydroxyplatanic acid which is now described for the first time in the literature.

General experimental procedure
The 1 H and 13 C-NMR experiments were recorded in a Bruker spectrometer ARX, operating respectively at 400 MHz for 1 H and 100 MHz for 13 C, using deutero pyridin

Extraction and isolation of the chemical constituents
The leaves (541 g) were extracted consecutively with hexane (3 x 1 L), dichloromethane (3 x 1 L) and methanol (3 x 1 L) with intervals of 3 days between each extraction at room temperature. After distillation of the solvents, it was obtained in each extract 3.9 g, 4.7 g and 5.1 g of solid residues. The residue obtained from the MeOH extraction (5.1 g) was submitted to droplet countercurrent chromatography, using