Triterpenic Acids from Eugenia moraviana

Um novo triterpeno caracterizado como ácido 6α-hidroxibetulínico foi isolado das folhas e caule da planta, vulgarmente conhecida no Brasil como Cambuí, Eugenia moraviana (Myrtaceae), juntamente com três outras substâncias conhecidas, identificadas como ácido platânico, ácido betulínico e β-sitosterol. Através da análise de espectros incluindo NOE e experimentos de RMN em duas dimensões foi realizada a atribuição inequívoca dos deslocamentos químicos de 1H e de 13C do ácido 6α-hidroxibetulínico(ácido-3β,6α-diidróxi-20(29)-lupen-28-óico) e do ácido platânico.


Introduction
Eugenia moraviana Berg.(Myrtaceae) is a tree that occurs in South America, mainly in Brazil, Argentina and Paraguai 1 .The leaves of some Eugenia species are used in folk medicine for several therapeutics finalities 2,3 .Compounds such as flavonoids, triterpenes, tannins and especially essential oils constituted of monoterpenes and sesquiterpenes have already been isolated from the genus Eugenia [4][5][6] .However, no reports were found on the Eugenia moraviana constituents.Our phytochemical studies on the leaves and stems of E. moraviana resulted in the isolation of a new lupane-type triterpenoid, which was characterized as 6α-hydroxybetulinic acid (1) together with three known compounds identified as platanic acid (2), betulinic acid and β-sitosterol 7 .Compound 2 and betulinic acid have been attracting much attention in natural products chemistry because they present biological activities, e.g.anti-HIV 8 , antitumor promoter 9 , antimalarial and anti-inflammatory activities 10,11 .Previous assignments of 13 C signals 8,12,13 led to divergence between the C-18 and C-19 chemical shifts of compounds 1 and 2 and also between the C-15 and C-21 chemical shifts of 2. In the present study we used techniques like COSY, HETCOR, HETCORLR and NOE difference, *e-mail: gjvidotti@uem.bracquired at 300 MHz ( 1 H), to report an unequivocal and complete 1 H and 13 C assignment of compounds 1 and 2. Peng and cols 14 reported the betulinic acid resonance assignment through a combination of high resolution NMR spectroscopy and a computer-assisted structure elucidation expert system.The authors run 13 C/DEPT, DQF COSY, HMBC, HMQC and NOESY spectra, acquired with a 720 MHz ( 1 H) spectrometer.

Structural elucidation and NMR signal assignments of 6α-hydroxybetulinic acid (1)
Compound 1, mp 285-288 °C; was isolated as white crystals.Its EIMS spectrum showed a molecular peak [M] +• at m/z 472 corresponding to the formula C 30 H 48 O 4 and other peaks at m/z 248, 203, 187 and 175, which are characteristic for a pentacyclic triterpene skeleton of the lupane series 15 .The IR spectrum showed absorptions due to hydroxyl (3454 cm -1 ), carboxyl (1685 cm -1 ) and exo-methylene (1639 and 879 cm -1 ) groups.The complete and unequivocal 1 H and 13 C chemical shifts assignments of 1 were assisted by DEPT, COSY ( 1 H x 1 H), HETCOR ( 13 C x 1 H) and HETCORLR ( 13 C x 1 H) spectra (Table 1).The 13 C NMR spectral data of 1 were compared with those from 6β-hydroxybetulinic acid 16 .According to our results the C-17 was erroneously assigned and the C-15/C-21 and C-18/C-19 were interchanged.The antiperiplanar coupling (J 6,7a and J 6,5a ) of 10.4 Hz and the synclinal coupling (J 6,7e ) of 3.8 Hz for H-6 proton at δ 4.35, indicated that the hydroxyl group at C-6 is α-orientation.The NOE difference NMR experiment was also performed to confirm the hydroxyl group orientation at C-6. Irradiation of the H-6 signal at δ 4.35 produced an enhancement in the methyl protons resonances at δ 1.45 (H-24, 5.0%), 0.96 (H-25, 6.5%) and 1.19 (H-26, 5.9%), which reveals a coaxial relationship between H-6, H-24, H-25 and H-26 thus confirming the equatorial position of the hydroxyl group at C-6.According to the observed NOE enhancements the structure of compound 1 should be as shown in Figure 1.

Assignments of C-15, C-18, C-19 and C-21 in the platanic acid (2)
To unequivocally assign carbons 15, 18, 19 and 21 in compound 2 the NOE difference spectra and a HETCOR spectrum were obtained.Initially the proton at δ 3.71 was irradiated and a NOE enhancement was observed at δ 2.24 (H-29)..

O
Interpretation of the observed NOE enhancements suggests a structure for 2 as shown in Figure 2 that shows all protons mentioned above in 1,3-diaxial interaction.
After the interpretation of the NOE enhancements we were able to identify carbons 18 and 19 in the HETCOR spectrum.The proton at δ 2.40 (H-18) correlates with the carbon at δ 49.9, and the proton at δ 3.71 (H-19) correlates the carbon at δ 52.1.To assign carbons C-15 and C-21, the proton at δ 2.53 (H-13) was irradiated and the NOE enhancement was observed on the protons at δ 1.57 (Ha-15) and 1.03 (H-26).Those protons are on 1,3-diaxial positions with respect to H-13 (see Figure 2).The HETCOR spectrum indicated the correlation between the proton at δ 1.57 (H-15) and the carbon at δ 28.8.The carbon at δ 30.3, that showed no correlation, must be C-21.
The complete assignments for the 1 H and 13 C NMR signals, as well the 1 H x 1 H and 13 C x 1 H correlations of 2 are reported in Table 2. pyridine-d 5 with TMS as internal reference; CC: silica gel 60 (70-230 mesh) and silica gel 60 (230-400 mesh); TLC: silica gel F254 (0.25 mm in thickness).

Plant material
Leaves and stems of the plant were collected in September 1996, at the edge of Paraná river, Porto Rico city, State of Paraná, Brazil and identified by Dr. Graziela Maciel Barroso, Jardim Botânico do Rio de Janeiro and Dr. Lucia Elena Soares e Silva, Universidade Federal de Brasilia.A voucher specimen (HUM 2163) was kept at the herbarium of the Biological Department of the Universidade Estadual de Maringá.

Figure 1 .
Figure 1.Schematic representation of selected NOE difference spectroscopy of 1.