The use of 13C and ¹H-NMR in the structural elucidation of a new Nor-lupane triterpene

Abstracts

The new nor-lupane triterpene, isolated from the leaves of Eugenia florida DC (Myrtaceae) was identified on the basis of its spectroscopic data, 13Cand¹H-NMR-2D, specially COSY¹H-¹H, COSY¹H-13C and HMBC correlations, as 28-O-beta-D-glucopyranosyl ester of 29-hydroxyplatanic acid.

Nor-lupane triterpene; 13C and¹H-NMR; HMBC; COSY; Eugenia florida; Myrtaceae

Um triterpeno nor-lupânico, inédito, foi isolado das folhas de Eugenia florida DC (Myrtaceae). Tomando-se por base as evidências espectroscópicas, RMN ¹H,13C, experimentos de RMN 2D, em especial correlações homonuclear ¹H-¹H, heteronuclear¹H-13C e correlações a várias ligações (HMBC), sua estrutura foi elucidada como: 29-hidroxiplatan-28-ato de beta-D-glicosila.

Article

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

Mário J. Junges ^a , João B. Fernandes ^b *; Paulo C. Vieira ^a , Maria Fátima das G. Fernandes da Silva ^b , and Edson Rodrigues Filho ^b

aDepartamento de Química, Universidade Federal do Rio Grande, Rua Eng. Alfredo Huch 475, C.P. 474, 96201-900 Rio Grande - RS, Brazil

^b Departamento de Química, Univesidade Federal de São Carlos, Rod. Washington Luis km 235, 13565-905 São Carlos - SP, Brazil

Um triterpeno nor-lupânico, inédito, foi isolado das folhas de Eugenia florida DC (Myrtaceae). Tomando-se por base as evidências espectroscópicas, RMN ¹H, ¹³C, experimentos de RMN 2D, em especial correlações homonuclear ¹H-¹H, heteronuclear ¹H-¹³C e correlações a várias ligações (HMBC), sua estrutura foi elucidada como: 29-hidroxiplatan-28-ato de b-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, ¹³Cand ¹H-NMR-2D, specially COSY ¹H-¹H, COSY ¹H-¹³C and HMBC correlations, as 28-O-b-D-glucopyranosyl ester of 29-hydroxyplatanic acid.

Keywords: Nor-lupane triterpene, ¹³C and ¹H-NMR, HMBC, COSY, Eugenia florida, Myrtaceae

Introduction

The triterpenes are a large group of plant substances with a wide spectrum of biological activities, such as anti-HIV¹. The study of the Eugenia florida DC (Myrtaceae) leaves, which occur in the areas of tropical and subtropical² 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 In this work an unpublished triterpene with skeleton nor-lupane was identified.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 ¹H-¹H and one bond and multiple bond heteronuclear ¹H-¹³C 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 ¹H and ¹³C-NMR 1D and 2D data with those reported in the literature for platanic acid^1,3,4. In the ¹H-NMR spectrum of 1, it was observed the presence of 5 methyl signals, d 0.77, 0.85, 0.94, 0.96 and 1.07 (Table 1). The signal at d 2.21 of the CH₃-29, which is characteristic of platanic acid¹ was not detected. At d 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 multiplet appearing at d 3.36-3.43 corresponds to H-19, coupled with H-18 and H-21. The COSY ¹H-¹H spectrum of 1 (Fig. 1) also exhibited several important correlations to establish the triterpene skeleton and the attribution of the ¹H 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 CH₂-29 (d 68.6), whose ¹H signals appeared as two doublets at d 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 (d 1.07) and H-24 (d 0.85) with C-3 at d 78.1 (³J, Table 1), and with the carbons C-5 at d 55.7, and C-4 at d 39.2 (²J). It was also noticed the correlation of the methyl H-25 (d 0.77) with C-10 at d 37.4 (²J) and of two methyl groups, H-26 and H-27 with C-8 at d 40.9 (respectively ²J and ³J). H-29 showed correlation with the carbonyl at d 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 d 5.95 (d, J = 8.4 Hz), confirmed by other signals between d 3.70 and 4.36. The inspection of the COSY ¹H-¹H 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 d 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 b-D-glucose⁷. After having assigned the ¹H and ¹³C 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 ¹³C-NMR spectrum showed a carbonyl carbon signal at d 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 ¹H signal at d 5.95 was correlated to the carbonyl carbon at d 174.7 (³J). This unequivocally confirmed that the position C-28 was indeed esterified with b-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), 473 (20), 275 (32), 183 (100) Daltons.

These data allowed to attribute the structure of 1 as being 28-O-b-D-glucopyranosyl ester of 29-hydroxyplatanic acid which is now described for the first time in the literature.

Experimental

General experimental procedure

The ¹H and ¹³C-NMR experiments were recorded in a Bruker spectrometer ARX, operating respectively at 400 MHz for ¹H and 100 MHz for ¹³C, using deutero pyridin (C₅D₅N) as solvent, with TMS as internal standard. HMQC and HMBC data were acquired using the microprogram invbtp (J = 145 and 9 Hz respectively). Melting point: Kofler block (Rochester) with one non calibrated thermometer. FT/IR: Bomen spectrometer, model M 102, KBr (1% of sample). For the chromatographic analysis a droplet countercurrent chromatograph Eyela DCC-300 model, equipped with 300 columns (42.0 x 0.3 cm) and also a R-HPLC Shimadzu, model CR4A equipped with UV detector Shimadzu model SPD-6AV were used.

Plant material

Leaves of Eugenia florida DC, were collected in February of l991, in the Ecological reserve of the Luís Antônio - São Paulo, Brazil and identified by Drs. José Rubens Pirani (USP) and Marcos Sobral (UFRGS). The respective voucher was deposited in the herbarium of the Instituto de Biociências of University of São Paulo-SP (SPF 75745). The leaves were dried separately in a stove with circulating air at 40 °C and grounded in a Willey mill.

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 CHCl₃:MeOH:H₂O (5:5:3 v/v) as solvent, with the organic layer being the mobile one. After 48 h of analysis, 200 fractions, 13 mL each, were collected and combined into 8 groups based on the results from analytical TLC. The fraction 67-74 (15 mg) was crystallized in methanol:acetone (9:1) and submitted to recycling-HPLC. Solutions of 7.5 mg mL^-1 in MeOH were prepared and 2 mL of these solutions were chromatographed on polymeric packing column (Asahipak GS-310 P, 21.5 cm ID x 50.0 cm L) using MeOH for elution with a flow rate of 8 mL min^-1. The UV detector was set at 215 nm. Three cycles of 60 min afforded compound 1 (9 mg, 28-O-b-D-glucopyranosyl ester of 29-hydroxyplatanic acid), solid, mp 240-241 °C. IR n_max KBr disc cm^-1: 3398, 2944, 2869, 1744, 1714, 1459, 1378, 1065, 589. ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz): Table 1. FAB/MS, m/z (relative intensity, %): 637 (M+1, 3), 636 (5), 473 (20), 275 (32), 183 (100).

Acknowledgement

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES), MCT/PRONEX Financiadora de Estudos e Projetos (FINEP) for financial support and NPPN/UFRJ by FAB/MS.

Received: March 29, 1999

FAPESP helped in meeting the publication costs of this article

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Publication Dates

• Publication in this collection
  21 June 2001
• Date of issue
  Aug 1999

History

• Received
  29 Mar 1999