Complete Assignments of ¹H and 13C-NMR Spectra of the 3,4-seco-Triterpene Canaric Acid isolated from Rudgea jasminoides

Note

Complete Assignments of ¹ H and ¹³ C-NMR Spectra of the 3,4- seco -Triterpene Canaric Acid isolated from Rudgea jasminoides

Marcia N. Lopes^a, Fabiano C. Mazza^a, Maria Claudia M. Young^b, and Vanderlan da S. Bolzani^a*

^a Instituto de Química, Universidade Estadual Paulista, C.P. 355, 14800-900 Araraquara - SP, Brazil

^b Seção de Fisiologia e Bioquímica de Plantas, Instituto de Botânica, C.P. 4005, 01061-970 São Paulo - SP, Brazil

O ácido canárico 1 foi isolado das folhas de Rudgea jasminoides. A substância isolada é um derivado triterpênico do tipo seco-lupano e teve sua estrutura elucidada com base nos dados espectrais, principalmente em experimentos de RMN a 1D e 2D. O sitosterol, o estigmasterol e os ácidos ursólico e oleanólico também foram isolados.

The canaric acid 1 was isolated from the leaves of Rudgea jasminoides. Compound 1 is a seco-lupane derivative and its structure was established on the basis of spectral data, mainly by 1D and 2D NMR experiments. Sitosterol, stigmasterol and ursolic and oleanolic acids were also isolated.

Keywords: Rudgea jasminoides, Rubiaceae, seco-A-ring lupane triterpene

Introduction

The genus Rudgea is widely distributed along the Brazilian East Coast, where some species are used as a remedy to treat rheumatism, syphilis and swelling of the members relief¹. Rudgea jasminoides is a small tree, especially impressive due to the pleasant jasmine smell of its wonderful white flowers, during the blooming. Several triterpenes and saponins have been isolated from other species, R. viburnioides². However, seco-lupane triterpene derivatives have never been isolated from the Rubiaceae family.

As a part of our study on the constituents of plants from Rubiaceae^2-5, we report the isolation of the canaric acid 1 from the leaves of a specimen of R. jasminoides. The canaric acid is a rare seco-ring-A-triterpene derivative, previously isolated from Dacryoides edulis, Canarium zeylanicum and C. muelleri from the Burseraceae family^6,7,8. However, no detailed studies of ¹³C-NMR, and high-resolution ¹H-NMR spectra of the compound 1 have been carried out. Herein we report application of these NMR spectral data in the structural elucidation of this triterpene. In addition, sitosterol, stigmasterol and two known triterpenes ursolic and oleanolic acid were isolated.

Experimental

IR spectra were obtained on a Perkin-Elmer Model 1600 spectrometer; ¹H-NMR (200, 300 and 500 MHz) and ¹³C-NMR (75 and 125 MHz) spectra were registered on a Bruker AC 200, DPX 300 and ARX 500, at 25°, in CDCl₃; HRFABMS: Central Analítica, Instituto de Química, Universidade de São Paulo, SP, Brasil.

Plant material

Leaves of Rudgea jasminoides were collected from preserved areas of Atlantic forest in the biological reserve of the “Ilha do Cardoso”, Cananéia, SP, Brazil. A voucher specimen SP 161 348 was deposited in the Herbarium Maria Eneida de Fidalgo, Instituto de Botânica de São Paulo.

Extraction and isolation of the constituents

Dried and powdered leaves (1.0 kg) of R. jasminoides were successively extracted with acetone and EtOH at room temp. to yield the acetonic (11.0 g) and EtOH (3.0 g) extracts respectively. Half of the acetone extract (5,5 g) was submitted to flash column chromatography eluting with a mixture of hexane and EtOAc. Twenty fractions (25 mL) were collected and analyzed by TLC on silica gel in hexane-EtOAc (7:3) and CHCl₃-MeOH (9:1 and 9.5:0.5). Fractions 2-3 were submitted to prep. TLC (hexane-EtOAc, 7:3) yielding sitosterol (21 mg) and stigmasterol (5 mg). Fractions 12-16 (200 mg) containing the crude triterpene 1 as a mixture with oleanane and ursane acid were further purified by a sequence prep. TLC with hexane-EtOAc (7:3) and CHCl₃-MeOH (9:1) to give compound 1 (43 mg) and oleanolic and ursolic acids (102 mg) in a mixture (2:1). The EtOH extract was diluted with H₂O and extracted with CHCl₃ at room temp. The CHCl₃ soluble portion was chromatographed on a silica gel column with a mixture of CHCl₃-MeOH. From the CHCl₃-MeOH (98:2) eluate was obtained a mixture of triterpenes and steroids. This mixture furnished the compounds sitosterol in a mixture with stigmasterol (12 mg) and ursolic acid (9 mg) by repeated column chromatography and prep. TLC.

Canaric acid ( 1 )

Colorless crystalline powder, mp. 215-217^o [lit.(8) mp. 215-217^o, [a]_D²⁵ +43^o (0.5, CHCl₃) [lit.(8) [a]_D²⁵ +57]; IR n_max cm^-1: 1690, 1646, 895, 880; FABMS m/z: 463 [M+Na]⁺; ¹H-NMR (CDCl₃): Table 1. ¹³C-NMR (CDCl₃): Table 1.

Methylation of canaric acid (1)

Compound 1 was methylated with CH₂N₂, affording a methyl canarate (1a). Gum, [a]_D²⁵ +038 (0.6, CHCl₃); IR n_max cm^-1: 1715, 1640, 1100, 880; FABMS m/z: 477 [M+Na]⁺; ¹H-NMR (CDCl₃) d: 4.76 (1H, s, H_a-24), 4. 60 (1H, s, H_a-29), 4.55 (1H, s, H_b-24), 4.50 (1H, s, H_b-29), 3.53 (3H, s, OCH₃), 2.53 (1H, br s, H-18), 0,75 (3H, s, H-26), 0.78 (3H, s, H-27), 0.98 (3H, s, H-28), 1.10 (3H, s, H-25), 1. 65 (3H, s, H-H-30), 1.63 (3H, s, H-23); ¹³C-NMR (CDCl₃) d: 178.0 s (C-3), 150. 9 s (C-20), 148.1 s (C-4), 113.5 t (C-24), 109.0 t (C-29), 51.3 q (OCH3), 50.7 d (C-9), 48.6 d (C-18), 48.0 d (C-19), 42.9 s (C-17), 42.5 s (C-14), 40.9 d (C-5), 40.5 t (C-22), 40.0 s (H-8), 39.0 s (H-10), 38.5 d (H-13), 36.0 t (H-16), 33.9 t (C-7), 32.9 t (C-1), 29.9 t (C-21), 27.5 t (C-12), 27.0 t (C-15), 24.4 t (C-6), 23.6 q (C-25), 21.7 t (C-11), 20.0 q (C-23), 19.0 q (C-30), 18.3 q (C-28), 16.1 q (C-26), 14.3 q (C-27).

Results and Discussion

The acetone extract of the leaves of R. jasminoides was chromatographed on a silica gel flash column and the fractions containing the mixture of triterpenes were further purified by prep. TLC to obtain the triterpene 1. All compounds isolated are known and were identified by spectroscopic means and comparison with authentic samples.

Compound 1 gave a positive test with Liebermann Buchard and ceric sulfate. The FABMS of 1 revealed the peak [M+Na]⁺ at m/z 463 consistent with the same molecular formula C₃₀H₄₈O₂ (m/z 440 [M]^+.) of canaric acid^6,7,8. The assignments of the NMR data of 1 and 1a were confirmed using a combination of DEPT, COSY, HMBC and NOESY experiments and comparison with data of known lupene and lupane derivatives^9-11. The ¹³C chemical shifts of 1 (Table 1) and 1a (see Experimental), especially those corresponding to C-20 and C-29 at d 150.78 and 109.50 (1) and 150.9 and 109.0 (1a), clearly corroborate the lupane structure of the compound 1. The combined ¹H and ¹³C-NMR data, together with DEPT and HMQC experiments (Table 1) suggested the presence of six tertiary methyl groups, two of them located at sp² carbons; twelve methylenes, five methines and seven quaternary carbons in the molecule of 1. The ¹H-NMR spectrum (Table 1) showed that 1 has six singlet methyls (d 0.72, .0.78, 0.95, 1.08, 1.61 and 1.65; two attributable to methyl groups at sp² carbons d 1.61 and 1.65) and four hydrogens typical of terminal double bonds at d 4.50, 4.57, 4.61 and 4.77. The ¹³C-NMR spectra suggested the presence of a carboxylic acid (d 179.91) and two terminal double bonds (d 150.78, 109.50 and 147.58, 113.38 respectivelly). ¹H and ¹³C-NMR spectra of the triterpene 1 resembled very closely those of lupeol⁹ and lupene¹⁰. Prominent differences in the ¹H and ¹³C values between lupeol and 1 were only observed in the A-ring region; the value of d 78.8 attributed to C-3 in lupeol^9,11 was inexistent in the molecule of 1. However, the signal at d 179.91 could be attributable to C-3, if we consider that the modification in the A-ring of the compound in discussion is related to the breaking of the C-3/C-4 bond (seco-lupane-A ring). These results allowed us to deduce that compound 1 possesses the same structure as canaric acid, previously described in the literature^6,7.

Interpretation of ¹H-¹H COSY, HMQC, HMBC, NOE and NOESY data for 1 led to the unambiguous elucidation of the structure. The relative stereochemistry of 1 was assigned on the basis of coupling constants (Table 1) and NOESY data (Fig. 1). The a-axial orientation of H-5 was confirmed by a strong NOE between H-5 and the hydrogens assigned as H-1, H-6a, H-9a, H-7a and 3H-23, and the lack of an NOE between it and the olefinic hydrogens assigned as H-24. Also consistent with the axial orientation of H-5 was a strong correlation observed with the axial methyl hydrogens 3H-25 and olefinic hydrogens H-24. Strong NOE correlations between 3H-25 and H-11b, between 3H-26 and H-7b and H-13b, between 3H-27 and H-12a, H-16a and H-18a, between 3H-28 and H-19b were consistent with trans B/C/D ring junctions with seco-ring A in a diequatorial conformation and ring D in a half boat conformation. Other correlations observed in the NOESY and NOE difference spectra are depicted in Table 2 and with the aid of COSY spectra and coupling constant values the relative stereochemistry of the seco-lupane triterpene 1 was established and shown to be identical to dihydrocanaric acid isolated from Hoya naumanii¹².

Received: March 5, 1998

FAPESP helped in meeting the publication costs of this article

Dedicated to the memory of professor W. Saffioti.

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Publication Dates

• Publication in this collection
  23 Oct 2002
• Date of issue
  1999

History

• Received
  05 Mar 1998