Isoflavonoids and triterpenoids isolated from Pterodon polygalaeflorus

Article

Isoflavonoids and Triterpenoids Isolated from Pterodon polygalaeflorus

Délcio D. Marques^a, Maria Iracema Lacerda Machado^a, Mário Geraldo de Carvalho^b, Luiz Augusto da C. Meleira^b, and Raimundo Braz-Filho^c

^aDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, C.P. 12.200, 60.455-760 Fortaleza - Ce, Brazil

^bDepartamento de Química - ICE, Universidade Federal Rural do Rio de Janeiro, 23851-970 Soropédica-Itaguaí - RJ, Brazil

^cSetor de Produtos Naturais, Universidade Estadual do Norte Fluminense, 28015-620 Campos - RJ, Brazil

Received: April 30, 1997

Os isoflavonóides 6,7-dimetoxi-3’,4’-metilenodioxi-, 4’-hidroxi-3’,6,7-trimetoxi-, 3,4,6,7-tetrametoxi-, 7-hidroxi-6-metoxi-3,4-metilenodioxi-, 2’,6,7-trimetoxi-3’,4’-metilenodioxi-, 2’,3’,4’,7,7-pentametoxi- e 2’,4’,5,6,7-pentametoxiisoflavona, os triterpenóides lupeol e betulina e o ácido 4-metoxibenzóico foram isolados dos extratos acetônicos do alburno e do cerne de Pterodon polygalaeflorus. As estruturas destes produtos naturais foram caracterizadas por métodos espectrométricos, principalmente experiências de RMN 1D e 2D de hidrogênio e carbono-13, que foram também utilizados para a atribuição inequívoca dos deslocamentos químicos dos átomos de hidrogênio e carbono-13 dos isoflavonóides.

The isoflavonoids 6,7-dimethoxy-3’,4’-methylenodioxy-, 4’-hydroxy-3’,6,7-trimethoxy-, 3,4,6,7-tetramethoxy-, 7-hydroxy-6-methoxy-3,4-methylenodioxy-, 2’,6,7-trimethoxy-3’,4’-methylenedioxy-, 2’,3’,4’,6,7-pentamethoxy- and 2’,4’,5,6,7-pentamethoxyisoflavone, together with the triterpenoids lupeol and betulin and 4-methoxybenzoic acid, were isolated from acetone extracts of the sapwood and heartwood of Pterodon polygalaeflorus. The structures of these natural products have been characterized by spectrometric methods, mainly extensive 1D and 2D NMR experiments, which were also used for complete assignments of the chemical shifts of the hydrogen and carbon-13 atoms of isoflavonoids.

Keywords: Pterodon polygalaeflorus, Leguminosae, isoflavonoids, triterpenoids, ¹H- and ¹³C-NMR data

Introduction

In a paper published recently the isolation of diterpenes from fruits of a specimen of Pterodon ploygalaeflorus Benth, Leguminosae family, was reported¹. As part of our continuing chemical investigation of this plant we have investigated the sapwood and the heartwood. The isoflavones 1-7, p-methoxybenzoic acid (8), lupeol (9), and betulin (10), have been isolated from this plant material. The structures of these natural products were established by spectral data, mainly ¹H- and ¹³C-NMR including homonuclear and heteronuclear 2D experiments (2, 3a, 5 and 7) and NOE difference spectra (1, 2, 3a, 5 and 7), which were also used for complete assignment of the hydrogen and carbon-13 atom chemical shifts of the isoflavonoid 2, the acetyl derivatives 3a, 5 and 7.

Results and Discussion

The acetone extracts of heartwood and sapwood of Pterodon ploygalaeflorus were submitted to chromatography on silica gel column to afford the isoflavonoids 1-7, 4-methoxybenzoic acid (8), lupeol (2) and betulin (10). The isoflavones 1, 2, 5-7 were previously isolated from Milletia dura (1 and 5)², Pterodon pubescens (2, 5 and 7)^3,4 and Condyla africana (6)⁵. The triterpenoids 9 and 10 are frequently found in plants⁶. The structural characterization of these compounds was based on spectral data, notebly the ¹H- and ¹³C-NMR spectra, including homonuclear ¹H-x ¹H-COSY and heteronuclear ¹H- x ¹³C-COSY-¹J_CH and ¹H- x ¹³C-COSY-ⁿJ_CH (n = 2 and 3)] experiments and NOE difference spectra (¹H{¹H}-NOE) data for the isoflavonoids 1, 2, 3a, 5 and 7, in addition to corresponding ¹H-NMR spectral data reported in the literature ^2-5 (Table 2). To the best of our knowledge, the isoflavone 3 is hitherto unreported as a natural product and ¹³C-NMR spectral data were only found for 2 (Table 2)⁷.

The homonuclear ¹H- x ¹H-COSY, heteronuclear ¹H- x ¹³C-COSY-ⁿ_JCH (n = 1, spin-spin couplings between ¹³C and ¹H- atoms via one bond; n = 2 and 3, COLOC = correlation via long-range couplings) 2D shift-correlated NMR spectra⁸ of 2, 3a, 5 and 7 (Tables 1-3) and NOE difference spectra⁸ of 1, 2, 3a, 5 and 7 (Table 4) together with the application of the shift parameters and the observed multiplicities of the signals of the carbon atoms deduced by comparative analysis of the PND- and DEPT-¹³C-NMR spectra⁹, were also used for the complete assignment of the hydrogen and carbon-13 atom chemical shifts of 2, 3a, 5 and 7, and consequently of the other isoflavonoids isolated from Pterodon polygalaeflorus by comparison with data now unambiguously assigned (Tables 1-4).

Thus, a series of 2D NMR experiments led to the assignment of all ¹H- and ¹³C resonances for 2 and 3a (e. g.). In the ¹H- x ¹³C-COSY-¹J_CH spectra of 2 and 3a the connectivities between the protonated carbon atoms and the corresponding hydrogens that were observed are: CH-2 [2: d_C 151.77(d) and d_H 7.97 (s); 3a: d_C 152.49 (d) and d_H 7.97 (s)], CH-5 [2: d_C 104.35 (d) and d_H 7.63 (s); 3a: d_C 104.60 (d) and d_H 7.60 (s)], CH-8 [2: d_C 99.18 (d) and d_H 6.88 (s); 3a: d_C 99.41(d) and d_H 6.87 (s)], CH-2’ [2: d_C 112.13 (d) and d_H 7.25 (d, J = 1.8 Hz); 3a: d_C 113.55 (d) and d_H 7.34 (d, J = 1.6 Hz)], CH-5’ [2: d_C 110.75 (d) and d_H 6.93 (d, J = 8.3 Hz); 3a: d_C 122.80 (d) and d_H 7.07 (d, J = 8.2 Hz); shifted downfield by only 0.14 ppm [Dd_H = 7.07 (3a)-6.93 (2)] and by 12.05 ppm [Dd_C = 122.80 (3a)-110.75 (2) in the acetyl derivative (3a), as anticipated by shielding reduction of the mesomeric ortho-effect], CH-6’ [2: d_C 120.61 (d) and d_H 7.05 (dd, J = 8.3 and J = 1.8 Hz); 3a: d_C 120.66 (d) and d_H 7.00 (dd, J = 8.2 and J = 1.6 Hz)] and methoxy groups [2: d_C 56.50 and d_H 3.99, 56.16 and 3.99, 55.62 and 3.92, 56.02 and 3.88; 3a: d_C 56.38 and d_H 3.87, 56.24 and 3.98, 55.85 and 3.86] (Tables 1 and 2).

The chemical shift assignments of the quaternary carbon atoms were established by ¹H- x ¹³C-COSY-ⁿJ_CH (n = 2 and 3, COLOC) spectra . Thus, the spectrum of 2 showed long-range correlations (Table 3): C-3 (d_C 123.91) with H-2 (d_H 7.97, ²J_CH), H-2’ (d_H 7.25, ³J_CH) and H-6’ (d_H 7.05, ³J_CH); C-4 with H-2 (d_H 7.97, ³J_CH) and H-5 (d_H 7.63, ³J_CH); C-6 (d_C 147.36) with H-5 (d_H 7.63, ²J_CH), H-8 (d_H 6.88, ³J_CH) and MeO-6 (d_H 3.99, ³J_CH); C-7 (d_C 154.02) with H-8 (d_H 6.88, ²J_CH), H-5 (d_H 7.63, ³J_CH) and MeO-7 (d_H 3.99, ³J_CH); C-9 (d_C 151.89) with H-8 (d_H 6.88, ²J_CH), H-2 (d_H 7.97, ³J_CH) and H-5 (d_H 7.63, ³J_CH); C-10 (d_C 177.51) with H-5 (d_H 7.63, ²J_CH) and H-8 (d_H 6.88, ³J_CH); C-1’ (d_c 124.51) with H-2 (d_H 7.97, ³J_CH) and H-5’ (d_H 6.93, ³J_CH); C-3’ (d_C 148.33) with H-5’ (d_H 6.93, ³J_CH) and MeO-3’ (d_H 3.92, ³J_CH); C-4’ (d_C 148.65) with H-2’ (d_H 7.25, ³J_CH), H-6 (d_H 7.05, ³J_CH) and MeO-4’ (d_H 3.88, ³J_CH). The spectra of 3a, 5 and 7 (d_H 7.97, ³J_CH) revealed analogous results as shown in Table 3.

Homonuclear NOE difference (¹Hx{¹H}-NOE) spectra (Table 4) of compounds 1, 2, 3a, 5 and 7 contributed to the assignments (Tables 1 and 2). The EIMS and IR spectra were also used (vide experimental).

Experimental

General experimental procedures

Mps were determined on a Mettler PF-5 melting point analyser. IR spectra were recorded in KBr using a Perkin-Elmer 720 infrared spectrometer. UV spectra were recorded in MeOH on a Varian-UV/ VIS 634-5 spectrometer. ¹H- and ¹³C-NMR spectra were obtained on a Bruker AC-200 spectrometer with standard pulse sequences operating at 200 MHz and 50.3 MHz, respectively, except the ¹H- NMR of 4a and 6 which were recorded on a Varian EM-360 (60 MHz) and Varian XL-100 spectrometers, respectively. The chemical shift values are reported in d (ppm) and the coupling constants (J) are in Hz; carbon multiplicities were determined by DEPT experiments; ¹H- x ¹H- - COSY, ¹H- x ¹³C - COSY - ¹J_CH, ¹H- x ¹³C - COSY - ⁿJ_CH (n = 2 and 3, COLOC), NOE difference spectra NMR experiments were carried out using Bruker commercial microprograms. Low-resolution EIMS (70 eV) data were obtained on a GC/MS Finningan 3300F/ 9500 apparatus. Chromatography was performed using Merck Kieselgel 0.05-0.20 mesh and TLC with Merck Kieselgel 60 F₂₅₄. TLC plates were examined under UV illumination and after exposure to iodine vapour.

Plant material

A specimen of Pterodon polygalaeflorus was collected in Monte Alegre - Bom Jesus, Piauí State, Brazil and identified by Professor Afrânio Gomes Fernandes (Universidade Federal do Ceará, Fortaleza, Ceará, Brazil). A voucher specimen is deposited at the Herbarium Prisco Bezerra of the Departamento de Biologia - Universidade Federal do Ceará.

Isolation of pterodon polygalaeflorus constituents

Acetone extraction of the heartwood

Dried and powdered heartwood (4.4 Kg) was continuously extracted with hot acetone. Upon removal of the solvent a residue (189 g) remained. This residue was chromatographed on a silica gel (756 g) column giving fractions H-1 to H-5, in this order, by elution with n-hexane-CHCl₃ (1:1), CHCl₃, CHCl₃ - acetone (1:1), acetone and MeOH. Fraction H-2 (43 g, eluted with CHCl₃) was rechromatographed on a silica gel column using cyclohexane- CHCl₃ (1:1) and CHCl₃ -acetone (8:2, 7:3 and 1:1) as eluents to obtain fraction H-2a to H-2d, respectively. Chromatography of fraction H-2a furnished 1 (56 mg), H-2b afforded 1 (75 mg) and 7 (284 mg), H-2c yielded 1 (74 mg) and 2 (605 mg) and H-2d funished 2 (310 mg) and 5 (560 mg).

Fraction H-3 (20 g), was eluted with CHCl₃-acetone (1:1) and rechromatographed on a sílica gel column furnishing fractions H-3a to 3c, in this order, by elution with CHCl₃-EtOAc (9.5:0.05, 9:1 and 7.5:2.5). These fractions afforded 1 (61 mg), 2 (398 mg) and 5 (544 mg), 3 (200 mg), 6 (283 mg) and 4 as acetyl derivative [4a (100 mg), obtained by treatment with Ac₂O/Py], respectively, after rechromatographed on silica gel columns.

Acetone extraction of the sapwood

Dried and powdered sapwood (4.4 Kg) was continuously extracted with hot acetone. The residue (200 g) obtained was chromatographed on a silica gel column using CHCl₃, CHCl₃-acetone (1:1), acetone and MeOH as eluents to furnish fractions 5-1 to 5-4. Fraction 5-1 (10 g) was rechromatographed on a sílica gel column to give 1 (50 mg), 8 (20 mg), 9 (539 mg) and 10 (130 mg).

6,7-Dimethoxy-3’,4’-methylenedioxyisoflavone (1)

Colorless crystals from MeOH, m.p. 240-241 °C. Spectral data are in accordance with values described in the literature². ¹³C-NMR: Table 1. ¹H-NMR: Table 3. NOE difference spectra (¹H-{¹H}-NOE) data: Table 4.

3’,4’,6,7-Tetramethoxyisoflavone (2)

Colorless crystals from MeOH, m.p. 188-189 °C. Spectral data are in accordance with literature values^3,4. ¹³C-NMR: Table 1. ¹H- NMR: Table 2. Heteronuclear 2D ¹H- x ¹³C shift-correlated via long-range coupling (¹Hx¹³C-COSY-ⁿJ_CH, n = 2 and 3): Table 3. NOE difference spectra data: Table 4.

4-Hydroxy-3’,6,7-trimethoxyisoflavone (3)

Colorless crystals, m.p. 279-281 °C. IR n_max cm ^-1: 3240 (OH), 1620 (C=O), 1590, 1510 (aromatic). ¹H-NMR (60 MHz, CF₃COOH) d_H: 8.80 (s, H-2), 7.84 (s, H-5), 7.50 (s, H-8), 7.10 (m, H-2’, H-5’ and H-6’), 4.27(s, MeO), 4.27 (s, MeO) and 4.04 (s, MeO). EIMS m/z (rel. int.): 328 (100, [M]^.+), 313 (6, [M-Me^.]⁺), 285 (5, [M-Me^.-CO]⁺), 181 (8, 3b), 180 (7, 3c), 148 (19, 3d).

4’-O-Acetyl-3’,6,7-trimethoxyisoflavone (3a)

Treatment of the isoflavone 3 (100 mg) with (Ac₂O) (2 mL) and pyridine (2 mL) at room temperature for 24 h, and usual work-up, produced 3a (98 mg), colorless crystals, m.p. 116-118 °C. IR n_max cm ^-1: 1760 (ester), 1625 (C=O), 1600, 1510 (aromatic). EIMS m/z (rel. int.): 370 (3, [M]^.+), 328 (100, [M-CH₂C=O]^.+), 327 (20, [M-Ac^.]⁺), 181 (3, 3b), 180 (4, 3c). Heteronuclear 2D ¹H- x ¹²C shift-correlated via long-range compling (¹Hx¹³C-COSY-ⁿJ_CH, n = 2 and 3): Table 3. ¹³C-NMR: Table 1. ¹H-NMR: Table 2. NOE dfference spectra (¹H-{¹H}-NOE) data: Table 4.

Methylation of 3

A solution of 3 (100 mg) in anhydrous acetone (40 mL) was treated with Me₂SO₄ (0.5 mL) in the presence of calcinated K₂CO₃, under reflux during 24 h. After filtration, the acetone was evaporated and the residue washed with 50% NH₄OH. The remaining residue was crystallized from MeOH to give 2.

6-Methoxy-7-0-acetyl-3’,4’-methylenedioxyisoflavone (4a)

Colorless crystals from MeOH, m.p. 204-205 °C. IR n_max cm^-1: 1740 (ester), 1650 (C=O), 1610, 1480 (aromatic). EIMSm/z (rel. int.) 354 (42, [M]^.+), 312 (100, [M-CH₂C=O] ^.+), 311 (20, [M-Ac]⁺), 166 (10, 4b), 146 (18, 4c). ¹H-NMR: Table 2.

2’,6,7-Trimethoxy-4’,5’-methylenedioxyisoflavone (5)

Colorless crystals from CHCl₃ + MeOH, m.p. 237-239 °C. Spectral data are in accordance with literature values^2-413C-NMR: Table 1. ¹H-NMR: Table 2. Heteronuclear 2D ¹H- x ¹³C shift-correlated via long-range coupling (¹H x ¹³C-COSY-ⁿJ_CH, n = 2 and 3): Table 3. NOE difference spectra (¹H-{¹H}-NOE): Table 4.

2’,4’,5’,6,7-Pentamethoxyisoflavone (6)

Colorless crystals from MeOH, m.p. 169-172 °C. Spectral data are in accordance with literature values⁵. ¹H-NMR: Table 2.

2’,3’,4’,6,7- Pentamethoxyisoflavone (7)

Colorless crystals, m.p. 170-172^oC. Spectral data are in accordance with literature values^3.4. ¹³C-NMR: Table 1. ¹H-NMR: Table 1. Heteronuclear ¹H- x ¹³C 2D shift-correlated via long-range coupling (¹Hx¹³C-COSY-ⁿJ_CH, n = 2 and 3): Table 3. NOE difference spectra (¹H-{¹H}-NOE) data: Table 4.

4-Methoxybenzoic acid (8, p-anisic acid)

Colorless crystals, m.p. 182-184 °C. (Lit.¹⁰ m.p. 184 °C). ¹H-NMR (60 MHz, CF₃COOH) d: 8.15 (d, J = 9.0 Hz, 2H-2,6), 7.08 (d, J = 9.0 Hz, 2H-3,5), 4.02 (s, MeO-4). EIMS m/z (rel. int.): 152 (98, [M]^.+), 135 (100, [M-OH]⁺), 107 (13, [M-OH-CO and/or M-COOH]⁺).

Lupeol (9)

Colorless crystals from MeOH, m.p. 211-214 °C. [Lit.¹¹ m.p. 215-216 °C (Me₂CO). Spectral data, mainly the chemical shifts and multiplicities of the signals of the carbon-13 deduced by comparative analysis of the PND- and DEPT-¹³C-NMR, and comparison with literature values¹² were used in the identification of this natural product.

Betulin (10)

Colorless crystals from MeOH, m.p. 249-251 °C. [Lit.¹³ m.p. 251-252 °C (EtOH)]. Spectral data, mainly the chemical shifts and multiplicities of the signals of the carbon-13 deduced by comparative analysis of the PND- and DEPT-¹³C-NMR, including the diacetyl derivative (10a) and comparison with literature values¹² were used in the identification of this compound.

Acknowledgments

This work was supported by CNPq fellowships and by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Programa de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT). The authors are also grateful to Professor Afrânio Gomes Fernandes, Universidade Federal do Ceará, for collection and identification of the plant material.

10. Reference 6: M-00529.

11. Reference 6: L-00498.

13. Reference 6: B-00951.

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Publication Dates

• Publication in this collection
  06 Nov 2002
• Date of issue
  May 1998

History

• Received
  30 Apr 1997