A new derivative of dihydroochnaflavone isolated from Luxemburgia species (Ochnaceae) and the complete ¹H and 13C NMR chemical shifts assignments

Um novo derivado da dihidroochnaflavona isolada de Luxemburgia species (Ochnaceae) e o completo assinalamento dos dados de RMN de ¹H e 13C

Abstracts

The biflavonoid 2",3"-dihydroochnaflavone, isolated from the Luxemburgia species (Ochanaceae), was treated with diazomethane to obtain the trimethyl-ether that was treated with pyridine/acetic anhydride to yield a new derivative, 5-acetil-7,4'-dimethyl-flavone-(3'→O-4'")-5"-acetil-7"-methyl-flavanone. The complete ¹H and 13C NMR data assignments of the new derivative were made by the one- and two-dimensional spectral analysis.

Ochnaceae; Luxemburgia; biflavone; trimethyl-diacetyl-dihydroochnaflavone

O biflavonóide 2",3"-diidroochnaflavona, isolada de espécies de Luxemburgia (Ochanaceae), foi tratada com diazometano e forneceu o éter trimetílico que, em seguida, foi tratado com anidrido acético/piridina obtendo-se um novo derivado, 5-acetil-7,4'-dimetil-flavona-(3'→O-4'")-5"-acetil-7"-metil-flavanona. Fez-se o completo assinalamento dos dados de RMN ¹H e 13C através da análise dos espectros uni- e bidimensionais do derivado.

Ochnaceae; Luxemburgia; biflavona; trimetil-diacetil-diidroochnaflavona

ARTIGO

A new derivative of dihydroochnaflavone isolated from Luxemburgia species (Ochnaceae) and the complete ¹H and ¹³C NMR chemical shifts assignments

Um novo derivado da dihidroochnaflavona isolada de Luxemburgia species (Ochnaceae) e o completo assinalamento dos dados de RMN de ¹H e ¹³C

Mario G. de Carvalho^*; Virginia C. Silva; José G. da Rocha Jr

Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, BR 465 KM 07, 23890-000, Seropédica-R.J, Brasil

ABSTRACT

The biflavonoid 2",3"-dihydroochnaflavone, isolated from the Luxemburgia species (Ochanaceae), was treated with diazomethane to obtain the trimethyl-ether that was treated with pyridine/acetic anhydride to yield a new derivative, 5-acetil-7,4'-dimethyl-flavone-(3'→O-4'")-5"-acetil-7"-methyl-flavanone. The complete ¹H and ¹³C NMR data assignments of the new derivative were made by the one- and two-dimensional spectral analysis.

Keywords: Ochnaceae, Luxemburgia, biflavone, trimethyl-diacetyl-dihydroochnaflavone.

RESUMO

O biflavonóide 2",3"-diidroochnaflavona, isolada de espécies de Luxemburgia (Ochanaceae), foi tratada com diazometano e forneceu o éter trimetílico que, em seguida, foi tratado com anidrido acético/piridina obtendo-se um novo derivado, 5-acetil-7,4'-dimetil-flavona-(3'→O-4'")-5"-acetil-7"-metil-flavanona. Fez-se o completo assinalamento dos dados de RMN ¹H e ¹³C através da análise dos espectros uni- e bidimensionais do derivado.

Unitermos: Ochnaceae, Luxemburgia, biflavona, trimetil-diacetil-diidroochnaflavona.

INTRODUCTION

The antitumor and cytotoxic activities of biflavonoids isolated from Luxemburgia species have been evaluated. Two natural biflavonoids 2",3"-dihydroochnaflavone and luxenchalcone was cytotoxic to murine Ehrlich carcinoma and human cells lines. The acetyl and the methyl derivatives were not cytotoxic to the analyzed tumour cells lines (Daniel et al., 2007; Oliveira et al., 2005). Species of Ochna, Ouratea and Luxemburgia (Ochnaceae) are great source of these natural biflavonoids (Likhitwitayawuid et al., 2001; Carvalho et al., 2002, 2004; Carbonezi et al., 2007). Besides the structural determination, we have analyzed the spectral data of the natural biflavonoids and their new derivatives (Carvalho et al., 2006a,b; Carvalho et al., 2003, Carvalho and Daniel, 2005). In this paper we describe the preparation and the complete proton and carbon-13 NMR assignments of a new derivative of 2",3"-dihydroochnaflavone.

MATERIAL AND METHODS

General procedures

Melting points are uncorrected. Fourier transform NMR spectra were recorded on a Varian INOVA-500 model spectrometer operating at 500 MHz for ¹H and 125 MHz for ¹³C. The NOE-difference experiment was recorded on a Bruker AC200 spectrometer operating at 200 MHz for ¹H. Samples were analyzed in 5 mm tubes, using CDCl₃ as solvent containing tetramethylsilane as internal standard. The natural biflavone (1) was isolated from the leaves of Luxemburgia nobilis and from L. octandra as described in the literature (Carvalho et al., 2002; Carvalho et al., 2004).

Preparation of 5,5"-diacetyl- 7,4',7"-trimethyl-2",3"-dihydroochnaflavone (1b)

The new derivative 1b was prepared by treating a methanol solution of the natural biflavone 1 (50.0 mg) with ethereal diazomethane solution prepared as the procedure described by Carvalho et al (2006b). After evaporation of the solvents, the residue was dissolved in acetone and purified by CC on silica gel. The fraction eluted with acetone yielded 1a (gum, 48.0 mg) identified as a trimethyl derivative. The product 1a was dissolved in 3.0 mL of Ac₂O:pyridine (1:1) solution and refluxed at 60 ºC overnight. Usual work-up yielded 1b (gum, 32.0 mg), Figure 1.

RESULTS AND DISCUSSION

The number of methoxyl groups detected by ¹H and ¹³C NMR spectra of the derivative 1a let us to confirm the dimmers structure. If it was a mixture of monomers (flavone and flavanone) this product would have five methoxyl groups. The analysis of ¹³C NMR (BBD and DEPT) spectra of compound 1b led us to identify twelve sp² CH including two signals at δ_CH 127.9 and 116.3, each representing two carbon atoms; signals of sp³ carbons at δ_CH 79.1 and δ_CH2 44.9 besides methoxyl and methyl groups; fourteen quaternary sp² carbons (4xC and 10xC-O) and four carbonyl groups at δ_C 188.7, 176.5, 169.5 and 169.4 were identified. In the ¹H NMR spectrum were identified ten protons of aromatic ring including two sets of meta-coupled doublets (¹Hx¹H-TOCSY) at δ_H 6.29 and 6.44 (J = 2.5 Hz) and at δ_H 6.65 and 6.87 (J = 2.0 Hz) which belong to the 6, 8, 6" and 8" protons of two flavonoid moieties (Table 1). The paramagnetic larger shift of the protons H-8/H-8" in comparison with H-6/6", reveals that, when the acetyl group is sterically hindered, the effect in para is more pronounced than in ortho effect in consequence of the shielding effect of the carbonyl group, the widespread statement when it is attributed major ortho effect of acetyl (Carvalho & Braz-Filho, 1993). The presence of a singlet at δ_H 6.76 (H-3) and the double doublets at δ_H 5.45 (J₁ = 13.5 and J₂ = 3.0 Hz; H-2"), 3.04 (J₁ = 16.5 and J₂ = 13.5 Hz; H-3"ax) and at δ_H 2.75 (J₁ = 16.5 and J₂ = 3.0 Hz; H-3"eq) were similar to those of 1 and 1a. The chemical shifts of ring B proton in the flavone moiety was identified by the signals at δ_H 7.13 (d, J = 8.7 Hz, H-5'), 7.58 (d, J = 2.0 Hz, H-2') and 7.75 (dd, J₁ = 7.8 and J₂ = 2,0 Hz, H-6') Table 1. The AA'BB' system was presented by two doublets (J= 8.5 Hz, 2H each) at δ7.42 and 7.0 which were assigned to H-2"',6"' and H-3"',5"' of the flavanone moiety, respectively. The cross peaks of heteronuclear long-range couplings observed in the ¹³Cx¹H-COSY-ⁿJ_CH (n = 2 and 3, HMBC) spectra of 1b were used to identify the chemical shift of quaternary carbons (Table 1). The signals of H-3 with C-10, C-4, C-2, C-1'; of H-6 with C-5, C-7 and C-10 besides signals of H-8 with C-7, C-9 and C-10 confirmed the chemical shifts of quaternary carbons of ring A of the flavone moiety (Table 1). The spectra obtained from NOEDIFF-NMR experiments of 1b showed NOE signals at 7.13 (d, H-5'), 6.29 (d, H-6), 6.44 (d, H-8), 6.65 (d, 2.0 Hz, H-6") and 6.87 (d, H-8") with irradiation at the methoxy groups; additional NOE signals at at 3.04 (H-3a), 2.75 (H-3b) and at 7.42 (H-2"',6"') with irradiation in the frequency of H-2" (δ_H 5.45) confirmed the AA'BB' system located in the flavanone moiety. These observations confirmed the C-3'→O-C-4'" connection between the flavone and flavanone moieties. The comparison of the ¹³C NMR spectral data with those of 1 (Carvalho et al., 2002), along with the analysis of the ¹³Cx¹H-COSY, ⁿJ_CH (n = 1, HMQC, n = 2 and 3, HMBC), allowed to define the structure and make the complete proton and carbon-13 NMR chemical shift assignments of the new biflavonoid derivative 5,5"-diacetyl-(3'-O-4"')-7,4',7"-trimethyl dihydroochnaflavone (1b, Table 1).

ACKNOWLEDGEMENTS

The authors are grateful to CNPq, FAPERJ and CAPES for a research fellowship and for financial supports and to Instituto de Química/UNESP/Araraquara for the 500 MHz NMR spectra.

Received 17 July 2008

Accepted 9 December 2008

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Publication Dates

• Publication in this collection
  11 Aug 2009
• Date of issue
  Mar 2009

History

• Accepted
  09 Dec 2008
• Received
  17 July 2008