A New Pentacyclic Tr iterpene Isolated from Myroxylon balsamum ( syn . Myroxylon peruiferum )

Myroxylon balsamum (L.) Harms (syn. Myroxylon peruiferum L.f.), family Fabaceae, is a large size tree, with white flowers, winged and aromatic fruits. Its dispersion is wide, being spread from the South of Mexico until the North of Argentina. In Brazil, its distribution includes a vast range of the forest region of the country, being very common in the States of Bahia, Paraná and Mato Grosso. It is known as “cabriuna” and “cabriúna vermelha” 1. The red-brown wood shows fine stripes, is a little rough and has a peculiar scent due to essential oil. It is a heavy wood and is resistant to deterioration, being widely used in carpentry. The hurtled log supplies a well-known exudate known as balsam of Peru or Tolu or a red oil which is quite rich in vanilla. The oil was used formerly in popular medicine as expectorant for breathing affections and as a sedative in cases of cystitis. Now its use is limited to the perfumery industry and as sedative pills for coughs 1-2. Chemically, the balsam is a mixture of free acids, especially benzoic and cinnamic, and benzyl benzoate. This plant also furnishes a resinous fraction containing monoterpenoids, sesquiterpenoids, alcohols and phenylpropanoids derivatives2. From trunk wood were isolated isoflavones, pterocarpan, coumestans, flavanone, isoflavanones and arylbenzofuran 3,4. In this paper we report the isolation and structural determination of a new pentacyclic triterpene 1 from the leaves of a specimen of Myroxylon balsamum (Fabaceae) collected in Espirito Santo State, Brazil. The structure was established by spectral analysis of 1 and its acetyl derivative 1a, mainly through 1H and 13C NMR spectra, including homonuclear 1H-1H COSY and heteronuclear 1Hdetected (inverse method) 1H-13C HMQC 1JCH and 1H-13C HMBC nJCH (n=2 and 3) 2D shift-correlated spectra 5.


Introduction
Myroxylon balsamum (L.) Harms (syn.Myroxylon peruiferum L.f.), family Fabaceae, is a large size tree, with white flowers, winged and aromatic fruits.Its dispersion is wide, being spread from the South of Mexico until the North of Argentina.In Brazil, its distribution includes a vast range of the forest region of the country, being very common in the States of Bahia, Paraná and Mato Grosso.It is known as "cabriuna" and "cabriúna -vermelha" 1 .
The red-brown wood shows fine stripes, is a little rough and has a peculiar scent due to essential oil.It is a heavy wood and is resistant to deterioration, being widely used in carpentry.The hurtled log supplies a well-known exudate known as balsam of Peru or Tolu or a red oil which is quite rich in vanilla.The oil was used formerly in popular medicine as expectorant for breathing affections and as a sedative in cases of cystitis.Now its use is limited to the perfumery industry and as sedative pills for coughs [1][2] .Chemically, the balsam is a mixture of free acids, especially benzoic and cinnamic, and benzyl benzoate.This plant also furnishes a resinous fraction containing monoterpenoids, sesquiterpenoids, alcohols and phenylpropanoids derivatives 2 .From trunk wood were isolated isoflavones, pterocarpan, coumestans, flavanone, isoflavanones and arylbenzofuran 3,4 .
In this paper we report the isolation and structural determination of a new pentacyclic triterpene 1 from the leaves of a specimen of Myroxylon balsamum (Fabaceae) collected in Espirito Santo State, Brazil.The structure was established by spectral analysis of 1 and its acetyl derivative 1a, mainly through 1 H and 13 C NMR spectra, including homonuclear 1 H-1 H COSY and heteronuclear 1 H detected (inverse method) 1 H- 13 C HMQC 1 J CH and 1 H- 13 C HMBC n J CH (n=2 and 3) 2D shift-correlated spectra 5 .

Results and Discussion
The pentacyclic triterpene 1 was obtained as colorless crystals whose molecular formula C 31 H 52 O 2 (6 unsaturations) was deduced by comparative analysis of 13 1).Other heteronuclear long-range couplings are summarized in Table 1.
The position of H-11 in 1 [δ H 3.84 (dd, J = 3.5 and 8.9 Hz)] was defined as axial (H-11β) on the basis of its coupling constant with H-9 (δ H 1.69, d, J = 8.9 Hz).The equatorial orientation of the hydroxyl group at CH-3 (H-3 axial) was deduced by comparative analysis of the chemical shifts corresponding to CH 2 -1 (δ C 39.26), CH 2 -2 (δ C 27. 1 H and 13 C NMR assignments (Table 1) of the pentacyclic triterpene 1 were determined on the basis of HMQC  9 , and 3 (δ C 68.4) 7 were also used in this analysis.Thus, the structure of the new triterpenoid isolated from Myroxylon balsamum was established as 3β-hydroxy-11αmethoxyolean-12-ene (1).The prominent peaks at m/z 255 (60 %) and 271 (16 %) observed in the mass spectrum, which could result from cleavage involving the rings B and D, respectively, in accordance with the formation of a diene after MeOH elimination 10 , were also used in this structural elucidation.To the best of our knowledge, this triterpene 1 is hitherto unreported.

Experimental
General Experiments Procedures EIMS were recorded on a VG Platform II mass spectrometer. 1H (400 MHz), 13 C (100 MHz), 1 H- 13 C HMQC 1 J CH and 1 H- 13 C HMBC n J CH (n=2 and 3 spectra were recorded using on a Bruker ARX-400 spectrometer, in CDCl 3 as solvent; residual CHCl 3 (δ H 7.24) and the central peak of the triplet of CDCl 3 (δ C 77.00) were used as internal references.The multiplicities of the carbon signals were deduced by comparative analysis of the HBBD-and DEPT-13 CNMR spectra.Heteronuclear 1 H and 13 C connectivities were deduced by 1 H- 13 C HMQC 1 J CH [spin-spin coupling of carbon and hydrogen via one bond ( 1 J CH 145.0 Hz)] and 1 H- 13 C HMBC n J CH [n=2 and 3, spin-spin interaction of carbon and hydrogen via two ( 2 J CH ) and three ( 3 J CH ) bonds, optimized for n J CH of 9 Hz].IR spectra with KBr plates were obtained on a FTIR Perkin-Elmer 1600/1605 spectrometer.

Plant material
The leaves of Myroxylon balsamum were collected at Reserva Florestal de Linhares, Companhia Vale do Rio Doce (CVRD), Espirito Santo State, Brazil, during September 1996.A voucher specimen has been deposited in the CVRD Herbarium (voucher no CVRD-483).

Extraction and Isolation
The air dried and powdered leaves (328.0 g) of Myroxylon balsamum were successively extracted with hexane, EtOAc and MeOH at room temperature and the solvents removed under vacuum to yeld 36.1g(rich in waxes and carotenoids), 17.4 g and 5.25 g of residues, respectively.The residue (17.4) obtained from the EtOAc solution was chromatographed on a of silica gel column eluting with hexane/CH 2 Cl 2 mixtures of increasing polarity; a total of 45 fractions (ca. 100 mL each one) were collected and combined of the basis of TLC comparison.The fractions 18-22, eluted with hexane-CH 2 Cl 2 (1:1), furnished 1 (92.0 mg) after recristalization from CHCl 3 .
13hemical shifts (δ H and δ C ) and1H coupling constants (J in Hz, in parenthesis) obtained from the one dimensional 1 H and 13 C NMR spectra.Multiplicity of signals of carbon atoms deduced by comparative analysis of HBBD-and DEPT-13 C NMR spectra.Homonuclear 1 H-1 H COSY and heteronuclear 1 H-13C HMQC 1 J CH and 1 H-13C HMBC n J CH (n=2 and 3) 2D NMR spectra were also used for these assignments.3 OD), isolated from leaves of Eucalyptus camaldulensis var.obtusa (Myrtaceae)