Mono and diterpenes from seeds of Xylopia sericea

Resumo

A monoterpene, 3beta,6beta-dihydroxy-p-menth-1-ene has been isolated from the seeds of Xylopia sericea along with four kaurane, one beyerene, one atisene and four trachylobane diterpenoids, including the trachyloban-18- and 19-methyl esters. The X-ray crystal structure of methyl ent-trachyloban-18-oate was determined in order to make an unambiguous distinction between the 18- and 19-esters. The 13C NMR data for ent-15alpha-hydroxy-trachyloban-19-oic acid has been revised.

monoterpene; kaurane diterpenes; X-ray crystallography

monoterpene; kaurane diterpenes; X-ray crystallography

Artigo

MONO AND DITERPENES FROM SEEDS OF XYLOPIA SERICEA

Jacqueline A. Takahashi^#, Henriete S. Vieira, Maria Amélia D. Boaventura

Departamento de Química, ICEx, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte - MG

^# e-mail: [e-mail]

James R. Hanson, Peter B. Hitchcock

CPES, University of Sussex, BN1 9QJ Brighton - UK

Alaíde B. de Oliveira

Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Olegário Maciel, 2360, 30180-112 Belo Horizonte - MG

Recebido em 19/7/00; aceito em 9/1/01

A monoterpene, 3b,6b-dihydroxy-p-menth-1-ene has been isolated from the seeds of Xylopia sericea along with four kaurane, one beyerene, one atisene and four trachylobane diterpenoids, including the trachyloban-18- and 19-methyl esters. The X-ray crystal structure of methyl ent-trachyloban-18-oate was determined in order to make an unambiguous distinction between the 18- and 19-esters. The ¹³C NMR data for ent-15a-hydroxy-trachyloban-19-oic acid has been revised.

Keywords: monoterpene; kaurane diterpenes; X-ray crystallography.

INTRODUCTION

The genus Xylopia (Annonaceae) includes about 160 species and is a well-known source of kaurane and trachylobane diterpenoids¹. In our search for Brazilian plants rich in diterpenoids², which could be used in obtention of compounds with biological activity via microbial biotransformation³, we have carried out a phytochemical investigation of Xylopia sericea St. Hill. The composition of the essential oil of this plant has been analysed previously⁴. In this paper we report the isolation of ten diterpenoids^*, ent-kaur-16-en-19-oic acid (1), ent-15a-acetoxykaur-16-en-19-oic acid (2), ent-kauran-16b-ol (3), ent-16b-hydroxykauran-19-oic acid (4), methyl ent-15-atisene-19-oate (5) methyl ent-beyer-15-en-19-oate (6), and four diterpenes of the ent-trachylobane series, ent-15a-hydroxytrachyloban-19-oic acid (7), its acetylated methyl ester (8), ent-trachyloban-19-oic acid (9), its methyl ester (10) and methyl ent-trachyloban-18-oate (11).

In order to make an unequivocal distinction between compounds 10 and 11, the later was analysed by X-ray crystallography. A monoterpene, 3b,6b-dihydroxy-p-menth-1-ene (12) was also isolated.

EXPERIMENTAL

General experiments methods

¹H NMR spectra were determined at 360 or 400 MHz for solutions in deuteriochloroform or deutero-pyridin. ¹³C NMR and 2D NMR spectra were determined at 100 or 125 MHz. TMS was used as an internal standard. Mass spectra were determined at 70 eV. IR spectra were measured using KBr discs. TLC spots were detected by spraying with 2% ceric sulfate soln. in 50% sulfuric acid and heating at 100°C. Silica gel for chromatography was Merck 7734.

Extraction and isolation

Seeds (3 kg) of Xylopia sericea were collected in Caratinga, Minas Gerais State, Brazil. After dried, they were powdered and repeatedly extracted with hexane in a Soxhlet apparatus. The hexane extract (105 g) was subjected to column chromatography on silica gel in hexane by use of an increasing gradient of ethyl acetate and methanol. The less polar fractions containing crystalline material were further purified by column chromatography on silica using the same solvent system to give ent-kaur-16-en-19-oic acid (1, 1.11 g), ent-15a-acetoxy-kaur-16-en-19-oic acid (2, 0.42 g), ent-kauran-16b-ol (3, 0.015 g), and the major constituent ent-trachyloban-19-oic acid (9, 11.4 g). These compounds were identified by their m.p., IR and NMR data. One fraction containing a mixture of compounds was esterified with ethereal diazomethane and further submitted to column chromatography on silica (petrol/ethyl acetate 9:1) to give compounds methyl ent-15-atisene-19-oate (5, 0.045 g), methyl ent-beyer-15-en-19-oate (6, 0.021 g) and methyl ent-15a-acetoxytrachyloban-19-oate (8, 0.023 g). In another fraction it was detected, by NMR, together with 9, another compound that showed the same Rf value, both cocrystallized, the former being the major component of the mixture (c. 95%). After several unsuccessful attempts of purification by column chromatography (Si gel and Si gel impregnated with AgNO₃), this fraction was treated with ethereal diazomethane and the esters mixture was recrystallized with ethanol/chloroform (9:1). Two kind of crystals were detected and they were carefully separated by hand in accordance with their different morphology and successively recrystallized. Using this procedure, 0.82 g of methyl ent-trachyloban-19-oate (10) (plates) were obtained as well as 0.07 g of methyl ent-trachyloban-18-oate (11) (thin needles). The methyl ester 11, m.p. 107-109^oC (lit.¹⁰ 112^oC), was analysed by X-ray crystallography.

3b,6b-dihydroxy-p-menth-1-ene (12, 0.11 g) was isolated from the fractions eluted with hexane/ethyl acetate (1:1) and identified by its NMR spectra. The same fractions gave ent-16-b-hydroxykauran-19-oic acid (4, 0.11g) and ent-15a-hydroxytrachyloban-19-oic acid (7, 0.32g), the last as a powder, m.p. 265-267 ^oC (from EtOH), IR n_max/cm^-1: 3450, 3200, 2900, 1700, 1500, 1300, 1150, 700.

¹H NMR (C₅D₅N, 360MHz): d_H 0.62 ( 1H, m, H-12), 0.76 (1H, dd, J= 3.0 and 7.5 Hz, H-13), 0.97 (3H, s, H-20), 1.16 (H, s, H-17), 1.18 (3H, s, H-18), 3.39 (1H, s, H-15). ¹³C NMR (C₅D₅N, 125MHz): see Table 1. EIMS: m/z: 318 (M⁺, C₂₀H₃₀O₃), 300, 285, 261, 151, 123, 105, 91, 81, 41.

Crystallographic data and structure determination for compound 11 C₂₁H₃₂O₂ M_r 316.47, orthorhombic, space group P2₁2₁2₁ (No. 19), a=6.236 (2), b=11.658 (3), c=25.042 (8) A, a = b = g = 90^o, v=1820.5 (9) A³, Z=4, D_calc.=1.16 g.cm³, F(000) 696, monochromated Mo-Ka radiation l=0.71073 A, m = 0.07 mm^-1. Data were collected using a crystal of size 0.40 x 0.20 x 0.15 mm on an Enraf-Nonius CAd4 diffractometer. A total of 1872 reflections were collected for 2 < q < 25^o and 0 < h < 7, 0 < k < 13, 0 < l < 29. 1170 reflections with I > 2s (I) were used in the refinement. There was no crystal decay and no absortion correction was applied. The structure was solved by direct methods using SHELXS-86 and SHELX-93^6,7. The non-hydrogen atoms were refined anisotropically by full matrix least squares. Hydrogen atoms were included in riding mode with U_iso = 1.2U_eq(C) or 1.5U_eq (C) for methyl groups. The final R indices [I > 2s (i)] were R₁ = 0.058 and wR₂ = 0.120 and R indices (all data) were R₁ = 0.109, wR₂ = 0.146. The maximum shift/esd was 0.006. The tables of crystalographic data have been deposited with the Cambridge Crystallographic Data Centre.

RESULTS AND DISCUSSION

The hexane extract of the seeds of X. sericea was subjected to column chromatography on silica gel eluting with a gradient of solvents of increasing polarity from n-hexane and ethyl acetate to methanol. Further purification by silica column chromatography of the crude fractions led to the isolation of compounds 1-4, 7 and 9 (major constituent). One impure fraction was esterified with ethereal diazomethane to afford, after chromatography on silica gel column, compounds 5, 6 and 8. The spectroscopic data of these compounds are in accordance with the literature^8,9,10,11, but there were found some differences in the ¹³C NMR data obtained for compound 7, when compared with the work of Harrigan and collaborators¹². The trachylobane skeleton of compound 7 was confirmed by signals at d_H 0.62 and 0.76 (H-12 and H-13, respectively)⁹. The presence of a secondary alcohol was revealed by signals at d_H 3.39 and d_C 83.7 in the ¹H and ¹³C NMR spectra. The location of the hydroxyl group at C-15 was confirmed by the observation of an n.O.e. enhancement of H-15 (4.9 %) on irradiation of the H-17 methyl group resonance (d_H 1.16). This also gave an n.O.e enhancement to the cyclopropane signals (H-12 and H-13). The stereochemistry of this hydroxyl group was confirmed by the g-gauche shift for C-9. However, the position of the signal assigned to C-8 (reported d_C 46.0,¹² found 40.9) was different (see Table 1). Careful inspection of the ¹³C NMR spectrum for compound 7 revealed four quaternary non-oxygenated carbon signals at d_C 25.0, 37.9 (co-incident with a methylene signal), 40.9 and 43.6. These were assigned to carbons 16, 10, 8 and 4, respectively by comparison with the carbon assignments for ent-trachyloban-19-oic acid (9). We believe that the co-incident signal at d_C 37.9 may not have been noticed previously as the earlier assignment was carried out on a mixture of two diterpenes. In fact the data reported for C-8 of compound 6 might fit better for C-8 of the other component of the mixture, 16b-hydroxy-kaur-16-en-19-oic acid (4).

A fraction containing compound 9, together with a further compound with the same Rf value, was also obtained. Attempts at their separation by several chromatographic systems, including silica gel/AgNO₃ column chromatography, were not successful. Purification was only achieved after esterification followed by recrystalization from ethanol/chloroform (9:1) and careful manual separation of the two types of crystals present in this sample. This procedure was repeated several times to afford 820 mg of compound 10 (plates) together with another substance (compound 11), which crystallized as thin needles. The ¹H NMR spectrum of compound 11 contained three methyl signals at d_H 0.94, 1.11 and 1.12. The presence of an ester group was revealed by a signal at d_C 179.4 in the ¹³C NMR spectrum. A downfield shift for the signal assigned to C-4 led us to consider that the compound was the 4b-epimer of 10¹³. The configuration at C-4 was confirmed by the X-ray crystal structure of 11 (see Figure 1). A comparison of the ¹³C NMR data for compounds 10 and 11 is given in Table 1.

A monoterpene, identified as 3b,6b-dihydroxy-p-menth-1-ene (12) was isolated from the fractions eluted with hexane:ethyl acetate (1:1). Compound 12 was isolated previously from Eupatorium erythropappum¹⁴. The isolation of this monoterpene from Xylopia species has not been described previously.

ACKNOWLEDGMENTS

We thank CNPq and FAPEMIG (Brazil) for schollarships and financial support.

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*

Substituents which are above the plane of this paper are referred to as b and those which are below this plane are referred to as a. The function of the ent prefix is to invert the stereochemical descriptor in the name that follows. Systematically, then, these substituents are termed ent-a and ent-b respectively

⁵.

Datas de Publicação

• Publicação nesta coleção
  08 Nov 2001
• Data do Fascículo
  Out 2001

Histórico

• Aceito
  01 Set 2001
• Recebido
  19 Jul 2000