A new pentacyclic triterpene isolated from Myroxylon balsamum (syn. Myroxylon peruiferum)

Abstracts

The new pentacyclic triterpene, 11alpha-methoxy-beta-amyrin (1), was isolated from Myroxylon balsamum (L.) Harms (syn. Myroxylon peruiferum L.f.). Its structure was established on the basis of IR, MS, ¹H NMR, 13C NMR. 2D NMR experiments were also used to establish the structure and the hydrogen and carbon chemical shift assignments of the new triterpene.

Myroxylon balsamum (Myroxylon peruiferum); Fabaceae; 3beta-hydroxy-11alpha- methoxyolean-12-ene

Um novo triterpeno pentacíclico, o 11alfa-metoxi-beta-amirina (1) foi isolado de Myroxylon balsamum (L.) Harms (sin. Myroxylon peruiferum L.f.). A estrutura desta substância foi elucidada pela análise de seus dados de IV, EM, RMN ¹H e 13C. RMN bidimensional foi também utilizada para definir a estrutura e atribuir os deslocamentos químicos dos átomos de hidrogênio e carbono do novo triterpeno.

Short Report

A new pentacyclic triterpene isolated from Myroxylon balsamum (syn. Myroxylon peruiferum)

Leda Mathias^a, Ivo J. C. Vieira^a, Raimundo Braz-Filho^a* and Edson Rodrigues Filho^b

^aSetor de Química de Produtos Naturais, LCQUI, CCT, Universidade Estadual do Norte Fluminense, 28015-620, Campos - RJ, Brazil

^bDepartamento de Química, Universidade Federal de São Carlos, São Carlos - SP, Brazil

Um novo triterpeno pentacíclico, o 11a-metoxi-b-amirina (1) foi isolado de Myroxylon balsamum (L.) Harms (sin. Myroxylon peruiferum L.f.). A estrutura desta substância foi elucidada pela análise de seus dados de IV, EM, RMN ¹H e ¹³C. RMN bidimensional foi também utilizada para definir a estrutura e atribuir os deslocamentos químicos dos átomos de hidrogênio e carbono do novo triterpeno.

The new pentacyclic triterpene, 11a-methoxy-b-amyrin (1), was isolated from Myroxylon balsamum (L.) Harms (syn. Myroxylon peruiferum L.f.). Its structure was established on the basis of IR, MS, ¹H NMR, ¹³C NMR. 2D NMR experiments were also used to establish the structure and the hydrogen and carbon chemical shift assignments of the new triterpene.

Keywords:Myroxylon balsamum (Myroxylon peruiferum), Fabaceae, 3b-hydroxy-11a-methoxyolean-12-ene

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 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 ¹H detected (inverse method) ¹H-¹³C HMQC ¹J_CH and ¹H-¹³C HMBC ⁿJ_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 C₃₁H₅₂O₂ (6 unsaturations) was deduced by comparative analysis of ¹³C NMR (31 singlet signals) and DEPT-¹³C NMR data⁶ (Table 1) [q = 90^o : 6 CH, including one sp² (d_C 121.51, CH-12) and two sp³ oxygenated (d_C 78.61, CH-3, and 75.85, CH-11); q = 135^o : 9 CH₂ and 9 CH₃ including one methoxy group (d_C 53.67, MeO-11)]and LREIMS [m/z 424, 100% (M-MeOH]. The DEPT results [6 CH, 9 CH₂ and 9 CH₃ = C₂₄H₅₁] indicated that one exchangeable hydrogen (hydroxy group) was present, in accordance with the absorption at n_max 3380 cm^-1 observed in the IR spectrum and a monoacetyl [d_H 2.03 (s)] derivative (1a) obtained by treatment with Ac₂O in the presence of pyridine (see experimental). The ¹³C NMR spectra of 1 revealed signals at d_C 121.51 (CH-12) and 149.67 (C-13), indicating the presence of a double bond. Because the unsaturation number is 6, 1 must therefore be a pentacyclic triterpene.

The location of the methoxy group at CH-11 (d_C 75.85 and d_H 3.84) and not at CH-3 (d_C 78.61 and d_H 3.23) was defined by heteronuclear long-range couplings between CH-11 (d_C 75.85) and the methoxyl hydrogens (d_H 3.23, ³J_CH), CH-12 (d_C 121.51) and H-11 (d_H 3.84, ²J_CH) and C-13 (d_C 149.67) and H-11 (d_H 3.84, ³J_CH) observed in the HMBC spectrum (Table 1). Other heteronuclear long-range couplings are summarized in Table 1.

The ¹H NMR spectrum of 1 (Table 1) showed nine singlet signals corresponding to one methoxy [d_H 3.23 (MeO-11)] and eight tertiary methyl groups [d_H 1.21 (3H-27), 1.04 (3H-25), 1.00 (3H-23), 0.99 (3H-26), 0.91 (3H-29), 0.89 (3H-30), 0.83 (3H-28) and 0.80 (3H-24)].

The position of H-11 in 1 [d_H 3.84 (dd, J = 3.5 and 8.9 Hz)] was defined as axial (H-11b) on the basis of its coupling constant with H-9 (d_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 CH₂-1 (d_C 39.26), CH₂-2 (d_C 27.30), CH-3 (d_C 78.61), CH-5 (d_C 55.02), CH₃-24 (d_C 15.44) of 1 and the model triterpenoids 2 and 3 supporting a HO-3b (H-3a) and HO-3a (H-3b) [2 (HO-3b)/3 (HO-3a): d_C 39.5/33.5 (CH₂-1), 27.4/25.4 (CH₂-2), 78.7/76.0 (CH-3), 39.0/37.0 (C-4), 55.1/48.8 (CH-5) and 15.5/22.4 (CH₃-24)]⁷, since superimposition of the signals was observed for H-3 (d_H 3.23) and the methoxyl group (d_H 3.23) in the ¹H NMR. The shielding revealed by the chemical shifts corresponding to carbon signals of CH₂-1, CH-5 and CH₃-24 of 3 can be justified by g-effects attributed to the hydroxyl group at an axial position (H-3b equatorial). This deduction was confirmed by ¹H NMR and ¹H-¹H COSY spectra (200 MHz) of the monoacetyl derivative 1a, which revealed the signal corresponding to H-3 as a double doublet [J = 8.2 (axial-axial interaction) and 5.0 Hz] at d_H 4.49 [Dd_H = 4.49 (1a) – 3.23 (1) = 1.26 ppm].

¹H and ¹³C NMR assignments (Table 1) of the pentacyclic triterpene 1 were determined on the basis of HMQC and HMBC data and comparison to the known triterpene 2 [11a-hydroxy-b-amyrin, isolated from callus tissues of Stauntonia hexaphylla (Lardizabalaceae)]⁸. The ¹³C NMR spectra of 1 (Table 1), the 11-O-methyl ether of 2, showed signals that matched closely with those of the triterpene 2 (recorded in CDCl₃)⁸, revealing significant differences only in the chemical shifts of the methine CH-11 [d_C 75.85 (1) and 81.70 (2)] and the quaternary C-13 [d_C 149.67 (1) and 153.20 (2)] carbon atoms. These different chemical shifts may be justified by the presence of a hydrogen bond involving the hydroxy group at CH-11 of 2 and the solvent pyridine-d₅, and not in CDCl₃ as reported in the literature⁸. The ¹³C and ¹H chemical shifts of the methine CH-11 [d_C 76.6 and d_H 3.87 (dd, J = 3.5 and 8.6 Hz)] of camaldulensis acid (3b,30-dihydroxy-11a-methoxyurs-12-en-28-oic acid, 4, recorded in CD₃OD), isolated from leaves of Eucalyptus camaldulensis var. obtusa (Myrtaceae)⁹, and 3 (d_C 68.4)⁷ were also used in this analysis.

Thus, the structure of the new triterpenoid isolated from Myroxylon balsamum was established as 3b-hydroxy-11a-methoxyolean-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 elimination¹⁰, were also used in this structural elucidation. To the best of our knowledge, this triterpene 1 is hitherto unreported.

Other 11-hydroxy or 11-methoxy pentacyclic triterpenes described in the literature are 28-oic acids: 2a,3b,23-trihydroxy-11a-methoxyurs-12-en-28-oic acid [isolated from resin of Shorea robusta (Dipterocarpaceae)]¹¹, 11a-hydroxybetulinic acid [isolated from leaves of Licania pyrifolia (Crysobalanaceae)]¹² and 11a-hydroxytormentic acid [2a,3b,11a,19a-tetrahydroxyurs-12-en-28-oic acid, isolated from aerial parts of Rosa laevigata (Rosaceae)]¹³.

Experimental

General Experiments Procedures

EIMS were recorded on a VG Platform II mass spectrometer. ¹H (400 MHz), ¹³C (100 MHz), ¹H-¹³C HMQC ¹J_CH and ¹H-¹³C HMBC ⁿJ_CH (n=2 and 3 spectra were recorded using on a Bruker ARX-400 spectrometer, in CDCl₃ as solvent; residual CHCl₃ (d_H 7.24) and the central peak of the triplet of CDCl₃ (d_C 77.00) were used as internal references. The multiplicities of the carbon signals were deduced by comparative analysis of the HBBD- and DEPT-¹³CNMR spectra. Heteronuclear ¹H and ¹³C connectivities were deduced by ¹H-¹³C HMQC ¹J_CH [spin-spin coupling of carbon and hydrogen via one bond (¹J_CH 145.0 Hz)] and ¹H-¹³C HMBC ⁿJ_CH [n=2 and 3, spin-spin interaction of carbon and hydrogen via two (²J_CH) and three (³J_CH) bonds, optimized for ⁿJ_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/CH₂Cl₂ 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-CH₂Cl₂ (1:1), furnished 1 (92.0 mg) after recristalization from CHCl₃.

3b-Hydroxy-11a-methoxyolean-12-ene (1)

Colorless crystals, mp 172-173° C, [a]_D + 12.4° (c 0.73, CH₂Cl₂); IR (KBr) u_max 3380, 1461,1384, 1044, 915, 731 cm^-1; EIMS m/z (rel. int.) 456 (M^+. abs.), 425 (25, M-MeO^.), 424 (100, M-MeOH), 409 (51, M–MeOH-Me^.), 271 (16), 255 (60), 253 (6); ¹H (400 MHz, CDCl₃) and ¹³C (100 MHz, CDCl₃) NMR: Table 1.

3b-Acetoxy-11a-methoxyolean-12-ene

(1a, acetyl derivative of 1)

A sample of 1 (18.0 mg) was treated with Ac₂O (2.0 mL) and dry pyridine (1.0 mL) at room temperature overnight. After the usual work-up, the crude product was chromatographed on a silica gel column eluting with CHCl₃ to furnish the acetyl derivative 1a (16.0 mg) as colorless crystals, mp 149-151° C, [a]_D + 5.7° (c 0.965, CH₂Cl₂); EIMS (rel. int.) 498 (M^+. abs.), 466 (100, M-MeOH), 451 (8, M – MeOH - Me^.), 391 (14, M - MeOH - Me^. - AcOH), 255 (42), 253 (10); ¹H NMR (200 MHz, CDCl₃) d_H 5.30 (br s, H-12), 4.49 (dd, J = 8.2 and 5.0 Hz), 3.88 (dd, J = 8.8 and 3.6 Hz), 3.18 (s, MeO-11), 2.03 (s, AcO-3), 1.23 (s, 3H-23), 1.18 (s, 3H-14), 1.04 (s, 3H-25), 0.98 (s, 3H-26), 0.86 (s, 3H-27, 3H-28, 3H-29), 0.81 (s, 3H-30).

Acknowledgements

The authors are grateful to the Programa de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT)/Financiadora de Estudos e Projetos (FINEP) and Fundação Estadual do Norte Fluminense (FENORTE) for grants, to CNPq for a research fellowship, and to Dr. Vitor Rumjanek for reading the manuscript.

Received: February 18, 1999

FAPESP helped in meeting the publication costs of this article.

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Publication Dates

• Publication in this collection
  23 Oct 2000
• Date of issue
  Apr 2000

History

• Received
  18 Feb 1999