Further diterpenoids isolated from Pterodon polygalaeflorus

Abstracts

From fruits of Pterodon polygalaeflorus (Leguminosae) the new diterpene 6alpha-hydroxyvouacapane, the known diterpenoids vouacapane-6alpha,7beta,14beta,19-tetraol and methyl 6alpha,7beta-dihydroxyvouacapan-17b-oate and the flavonoid taxifolin were isolated. Structural determinations were accomplished by spectroscopic analysis, including two-dimensional NMR, chemical transformation of the new diterpene and comparison with literature data.

Pterodon polygalaeflorus; Leguminosae; diterpenoids; flavonoid

Dos frutos de Pterodon polygalaeflorus (Leguminosae) foram isolados o novo diterpeno 6alfa-hidroxivouacapano e os conhecidos diterpenóides 6alfa,7beta,14beta,19-tetraidroxivouacapano, 6alfa,7beta-diidroxivouacapan-17beta-oato de metila e o flavonóide taxifolina. As estruturas foram determinadas com base em análise espectroscópica, inclusive RMN bidimensional, transformação química do novo diterpeno e comparação com dados da literatura.

Short Report

Further diterpenoids isolated from Pterodon polygalaeflorus

Angela M. C. Arriaga ^a , Maria Augusta B. de Castro ^a , Edilberto R. Silveira ^a and Raimundo Braz-Filho ^b

^aDepartamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, CP 12200, 60451-970, Fortaleza, Ceará, Brazil

^b Setor Química de Produtos Naturais-LCQUI-CCT, Universidade Estadual do Norte Fluminense, 28015-620, Campos, Rio de Janeiro, Brazil

Dos frutos de Pterodon polygalaeflorus (Leguminosae) foram isolados o novo diterpeno 6a-hidroxivouacapano e os conhecidos diterpenóides 6a,7b,14b,19-tetraidroxivouacapano, 6a,7b-diidroxivouacapan-17b-oato de metila e o flavonóide taxifolina. As estruturas foram determinadas com base em análise espectroscópica, inclusive RMN bidimensional, transformação química do novo diterpeno e comparação com dados da literatura.

From fruits of Pterodon polygalaeflorus (Leguminosae) the new diterpene 6a-hydroxyvouacapane, the known diterpenoids vouacapane-6a,7b,14b,19-tetraol and methyl 6a,7b-dihydroxyvouacapan-17b-oate and the flavonoid taxifolin were isolated. Structural determinations were accomplished by spectroscopic analysis, including two-dimensional NMR, chemical transformation of the new diterpene and comparison with literature data.

Keywords: Pterodon polygalaeflorus, Leguminosae, diterpenoids, flavonoid

Introduction

In continuation of our systematic studies of the Brazilian northeast medicinal plants, we have undertaken a study of Pterodon polygalaeflorus Benth. (Leguminosae), which is used in folk medicine against bronchytis, amigdalytis and as tonic. We have already reported diterpenoids¹ and the volatile constituents² from this plant. In continuation to this phytochemical investigation, we now report the isolation and structure determination of a new diterpene 6a-hydroxyvouacapane (1) and of the known compounds vouacapane-6a,7b,14b,19-tetraol (2)³, methyl 6a,7b-dihydroxyvouacapan-17b-oate (3)⁴ and the flavonoid taxifolin (4, 5,7,3',4'-tetrahydroxydihydroflavonol)⁵ , isolated from the fruits. The structures of these compounds were deduced by spectral analysis, mainly ¹H and ¹³C NMR, including 2D-NMR experiments [¹H-¹H-COSY, ¹³Cx¹H-COSY-¹J _CH (HETCOR) and ¹³Cx¹H-COSY-ⁿJ _CH (n=2 and 3, COLOC)], chemical transformation of the new diterpene (1) and comparison with literature data.

Results and Discussion

The hot hexane extract of coarsely ground fruits of P. polygalaeflorus was submitted to aqueous ethanol alkaline hydrolysis. After usual work-up, the insaponifiable fraction obtained was chromatographed on silica gel column to furnish a new diterpene 6a-hydroxyvouacapane (1), methyl 6a,7b-dihydroxyvouacapan-17b-oate (2), previously isolated from Pterodon emarginatus⁴, vouacapane-6a,7b,14b,19-tetraol(3), which was also isolated from the seeds of a specimen of P. polygalaeflorus collected in Minas Gerais-Brazil⁶,and the flavonoid 4, known as taxifolin (5,7,3',4', tetrahydroxydihydroflavonol)⁵. Compound 1 was not previously obtained as a natural product, although it was prepared from its acetylated derivative 1a, which had been isolated from Dipterix lacynifera⁷.

The identification of the known compounds 2-4 involved the analysis of ¹H NMR (1D and 2D ¹H-¹H-COSY) and ¹³C NMR (HBBD and DEPT) spectral data (Table 1) and comparison with literature values^1,5,6. The comparative analysis of the chemical shifts of the hydrogen and carbon atoms of 2 and methyl 6a-acetoxy-7b-hydroxyvouacapan-17b-oate (2a) and 6a,7b-dihydroxyvouacapan-17b-oic acid (2b), which were also isolated from Pterodon polygalaeflorus¹, revealed, as anticipated, the modifications in the chemical shifts of the hydrogen H-6 [2: d_H 4.79 (dd, J=10.7, 8.9 Hz); 2a: d_H 5.25 (dd, J=11.5, 9.2 Hz)] and carbons C-5 [Dd_C = 55.8 (2) - 54.9 (2a) = 0.9 ppm], C-6 [Dd_C = 74.3 (2) - 75.5 (2a) = - 1.2 ppm] and C-7 [Dd_C = 82.8 (2) - 81.2 (2a) = 1.6 ppm] as a consequence of the absence of the g-effect (C-5 and C-7) and b-effect (C-6) of the carbonyl carbon of the 6a-acetoxy group in 2 (Table 1).

Compound 1 was obtained as colorless needles after crystallization from methanol. The HREIMS spectrum showed the molecular ion at m/z 302.2126 corresponding to the molecular formula C₂₀H₃₀O₂ (m/z 302.2238, Dm/z = 0.0112 daltons). This data together with the presence of signals for three quaternary [d_H 0.89 (s,3H-20), 1.07(s, 3H-19) and 1.17 (s, 3H-18)] and one tertiary [d_H 0.99 (d, J=6.9 Hz, 3H-17)] methyl groups, one carbinolic hydrogen at d_H 3.94 (td, J=7.1, 3.6 Hz, H-6) and two hydrogens at d_H 6.17 (d, J=1.6 Hz, H-15) and 7.20 (d, J=1.6 Hz, H-16) of a 2,3-disubstituted furan observed in the ¹H NMR spectrum allowed us to classify this diterpene as a vouacapanoid, the same class of diterpenoids which was reported previously¹. This deduction was confirmed by comparative analysis of the HBBD- and DEPT-¹³C NMR, which revealed the presence of signals corresponding to four quaternary (d_C 149.48, 122.14, 38.58 and 33.39), seven methine (d_C 140.42, 109.42, 70.02, 60.02, 44.46, 35.30 and 30.97), five methylene (d_C 43.69, 42.15, 39.81, 22.42 and 18.42) and four methyl (d_C 37.01, 22.57, 17.36 and 15.41) carbon atoms. The presence of one secondary hydroxyl group was indicated by ¹H NMR of the monoacetate derivative (1a), which revealed downfield shift for carbinolic hydrogen H-6 [Dd_H 5.12 (1a) - 3.93 (1) = 1.19 ppm].

The localization of the hydroxyl group at C-6 was deduced by the spin-spin interaction of hydrogen H-6 [d_H 3.94 (td, J=7.1, 3.6 Hz)] with H-5 [d_H 0.91 (d, J=7.1 Hz)] and 2H-7 (d_H 1.90 and 1.40) revealed by homonuclear 2D ¹H - ¹H-COSY spectrum and the chemical shift of C-5 (d_C 60.02) that was assigned on the basis of heteronuclear 2D direct CH [¹³Cx¹H-COSY¹J _CH: d_C 60.02 (C-5) correlated with H-5 (d_H 0.91)] and via long range coupling CH [¹³Cx¹H-COSY-ⁿJ _CH (n = 2 and 3) COLOC]: d_C 60.02 (C-5) correlated with 3H-18 (d_H 1.17)] spectra. Additional confirmation of the presence of a hydroxyl group at C-6 was obtained from the ¹³C NMR spectrum of the acetyl derivative of 1a: i) the signals of the carbon atoms C-5 (d_C 57.35) and C-7 (d_C 37.34) revealed, as anticipated, the g-effects of the carbonyl carbon of the acetoxyl group [C-5: Dd_C = 57.35 (1a) - 60,02 (1) = - 2.67 ppm; C-7: Dd_C = 37.34 (1a) - 42.15 (1) = - 4.81 ppm]; ii). The C-6 signal showed the downfield shift = Dd_C = 72.62 (1a) - 70,02 (1) = 2.6 ppm as a consequence of the b-effect of the carbonyl carbon atom (Table 2). The presence of a tertiary methyl group at C-14 [d_H 0.99 (d, J=6.9 Hz) and d_C 17.37] was confirmed by 2D carbon-hydrogen correlation (³J_CH) of the 3H-17 (d_H 0.99) with the carbon atom C-13 (d_C 122.14) observed in the ¹³Cx¹H-COSY-ⁿJ _CH (n = 2 and 3) spectrum.

The 2D carbon-hydrogen correlation experiments modulated with ¹J_CH established the hydrogen chemical shift correlations with those of the carbon atoms and also ²J_CH and ³J_CH couplings to detect hydrogen resonance correlated with not directly bound carbon signals, along with the application of the usual shift parameters and multiplicity of signals, were used to complete the assignment of the ¹H and ¹³C NMR spectra of 1 (Table 2).

The location of the hydroxyl group at chiral carbon C-6 in the equatorial position was deduced on the basis of the J-value (7.1 Hz) observed for the signal of the H-6 [d_H 3.94 (dd, J=7.1, 3.6 Hz)], downfield shift [Dd_H = 5.12 (1a) - 3.94 (1) = 1.18 ppm] observed in the ¹H NMR spectrum of the monoacetate 1a (Table 2). The monoacetate 1a, obtained by acetylation of 1 with Ac₂O/Py, was identified by a comparative study of the ¹H and ¹³C NMR spectra with the natural product isolated from Dipteryx lacunifera⁷.

Confirmation of 1 as a natural product (and not as a hydrolysis product) was obtained by preparation of a new hexane extract from fruits (1.5 kg) collected from the same specimen. The residue (16 g) was submitted to column chromatography on silica gel eluted with hexane, CHCl₃ and EtOAc. The residue (5.3 g) from the CHCl₃ elution was submitted to a preparative TLC affording 1 (12.7 mg).

The relative configuration of 1 was also deduced on the basis of the [a]_D= + 26.6 ± 0.05 (c 1.0, CHCl₃) compared with value described in the literature for this compound obtained of the acetyl derivative 1a by treatment with LiAlH₄⁷.

Experimental

General experimental procedures

Mps: uncorr. Optical rotation was obtained on a Perkin-Elmer 341 digital polarimeter; NMR: ¹H(200 MHz) and ¹³C (50.3 MHz) in CDCl₃ (1 and 2), CDCl₃ + acetone-d₆ (3 and 4) and TMS as int. standard; EIMS: 70 eV.; CC: silica gel (Merck 0.05-0.20 mm); TLC: silica gel H or G (Merck) and spots were visualized by exposure to iodine vapour.

Plant material

Pterodon polygalaeflorus Benth. was collected in May 1993 in Joaquim Pires, Piauí State, Brazil. A voucher specimen (N^o 8808) representing the collection was identified by Dr Afranio G. Fernandes (Botanist, Departamento de Biologia, UFC) and has been deposited at Herbario Prisco Bezerra of the Departamento de Biologia, Universidade Federal do Ceará, Brazil.

Extraction and isolation of the constituents

The powdered fruits (5 kg) were extracted with hexane in a Sohxlet apparatus to yield 500 g of a yellowish oil that was designated EH. The yellowish oil EH (100 g) was submitted to alkaline hydrolysis according to usual procedure, KOH-H₂O-EtOH (1:1) under reflux for 30 min. Column chromatography of the ppt (60 g) obtained after addition H₂O yielded 4 fractions designated EHH (8.6 g) eluted with hexane, EHC (17.6 g) eluted with CHCl₃, EHAE (21.3 g) eluted with EtOAc and EHM (10.0 g) eluted with methanol. Fraction EHAE (21.3 g) was chromatographed on a silica gel column: the fraction eluted with CHCl₃ yielded 6a-hydroxyvouacapane 1 (380 mg) after recrystallization from MeOH; the fraction eluted with CHCl₃-EtOAc (9:1) furnished methyl 6a,7b-dihydroxyvouacapan-17b-oate (2, 150 mg) after recrystallization from MeOH; the fraction eluted with CHCl₃-EtOAc (2:8) afforded vouacapane-6a,7b,14b,19-tetraol(3, 80 mg) after recrystallization from MeOH; and the fraction eluted with EtOAc yielded taxifolin (4, 5,7,3',4'-tetrahydroxydihydroflavonol, 100 mg) after recrystallization from MeOH.

6a-Hidroxyvouacapane (1)

M. p. 147.6-148.3 °C, needles from MeOH. [a]_D= + 26.6 ± 0.05 (c 1.0, CHCl₃). ¹H NMR (200 MHz, CDCl₃) and ³C NMR (50 MHz, CDCl₃): Table 2. HREIMS m/z (rel. int.): 302.2126 (C₂₀H₃₀O₂, m/z 302.2246, [M]^.+, 30); 284.2024 (C₂₀H₂₈O, m/z 284.2140, [M - H₂O]^.+, 89); 108.0550 (C₇H₈O, m/z 108.0575, 1b, 100).

Acetylation of 1

6a-Acetoxyvouacapane (1a) was obtained by treatment of 1 (105 mg) with Ac₂O (5 mL) and pyridine (1.5 mL). The usual work-up followed by CC on silica gel [hexane:CHCl₃ 1:1 as eluent] of the crude product furnished 1a (100 mg) as an oil fr. homogeneous by TLC. ¹H NMR (200 MHz, CDCl₃) and ¹³C NMR (50 MHz, CDCl₃): Table 2. HREIMS m/z (rel. int.): 344.2794 (C₂₂H₃₂O₃, m/z 344.2396, [M]^.+, 31); 316.2465 (C₂₁H₃₂O₂, m/z 316.2404, [M-CO]^.+, 10.5), 285.2315 (C₂₀H₂₉O, m/z 285.2220, [M-AcO^.], 20), (C₇H₈O, m/z. 108.0575, 1b, 47.5).

Acknowledgments

The authors are grateful to Brazilian agencies FINEP, CAPES, CNPq and FUNCAP for financial support and to Prof. Afrânio Gomes Fernandes (Universidade Federal do Ceará) for collection and identification of the plant material.

Received: February 22, 1999

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Publication Dates

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

History

• Received
  22 Feb 1999