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

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 relief. 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. viburnioides. 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, 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. However, no detailed studies of C-NMR, and high-resolution H-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


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 relief 1 .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. viburnioides 2 .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][3][4][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 13 C-NMR, and high-resolution 1 H-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.

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 hex-Note Dedicated to the memory of professor W. Saffioti.

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 C 30 H 48 O 2 (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][10][11] .The 13 C chemical shifts of 1 (Table 1) and 1a (see Experimental), especially those corresponding to C-20 and C-29 at δ 150.78 and 109.50 (1) and 150.9 and 109.0 (1a), clearly corroborate the lupane structure of the compound 1.The combined 1 H and 13 C-NMR data, together with DEPT and HMQC experiments (Table 1) suggested the presence of six tertiary methyl groups, two of them located at sp 2 carbons; twelve methylenes, five methines and seven quaternary carbons in the molecule of 1.The 1 H-NMR spectrum (Table 1) showed that 1 has six singlet methyls (δ 0.72, .0.78, 0.95, 1.08, 1.61 and 1.65; two attributable to methyl groups at sp 2 carbons δ 1.61 and 1.65) and four hydrogens typical of terminal double bonds at δ 4.50, 4.57, 4.61 and 4.77.The 13 C-NMR spectra suggested the presence of a carboxylic acid (δ 179.91) and two terminal double bonds (δ 150.78, 109.50 and 147.58, 113.38 respectivelly). 1 H and 13 C-NMR spec- atom