Flavones from the leaves of Ficus gomelleira

Article

Flavones from the Leaves of Ficus gomelleira

Daniel F. Amaral, Mara S. P. Arruda*, Alberto C. Arruda, Adolfo H. Müller, Luna L. J. Pantoja and Tânia M. da S. Lima

Departamento de Química, Centro de Ciências Exatas e Naturais, Universidade Federal do Pará, Campus Universitário, 66075-970, Belém - PA, Brazil

Duas novas flavonas, 5 -hidroxi -7,5' -dimetoxi -3',4' -metilenodioxiflavona e 5 -hidroxi -7,3',5' -trimetoxi -4' -(3,3 -dimetilaliloxi) flavona foram isoladas das folhas de Ficus gomelleira, juntamente com três flavonas conhecidas, a 5,6,7,3',4',5' -hexametoxiflavona, 5 -hidroxi -8,3',4' -trimetoxi -2'',2'' -dimetilpirano (5",6":6,7) -flavona e 5 -hidroxi -8,3',4',5' -tetrametoxi -2'',2'' -dimetilpirano (5",6":6,7) -flavona. As estruturas das substâncias isoladas foram elucidadas com base na análise de seus dados espectrais, bem como por comparação com dados da literatura.

Two new flavones, 5 -hydroxy -7,5' -dimethoxy -3',4' -methylenedioxyflavone and 5 -hydroxy -7,3',5' -trimethoxy -4' -(3,3 -dimethylallyloxy) flavone, as well as three known flavones: 5,6,7,3',4',5' -hexamethoxyflavone, 5 -hydroxy -8,3',4' -trimethoxy -2'',2'' -dimethylpyrano (5",6":6,7) -flavone and 5 -hydroxy -8,3',4',5' -tetramethoxy -2'',2'' -dimethylpyrano (5",6":6,7) -flavone were isolated from the leaves of Ficus gomelleira. Their structures were elucidated by spectroscopic methods and comparison with literature data.

Keywords:Ficus gomelleira, Moraceae, leaves, flavones

Introduction

In the course of our systematic studies on the species of genus Ficus of medicinal interest, which grows in the Amazon forest, we have investigated F. gomelleira. The leaves and latex of this species are used in folk medicine for intestinal diseases as laxative and vermifuge¹. Earlier studies on the genus Ficus (F. maxima and F. aripuanensis), carried out in our laboratory, have resulted in the isolation of flavonoids and terpenoids^2,3. From F. gomelleira two novel flavones, 5 -hydroxy -7,5' -dimethoxy -3',4' -methylene -dioxyflavone (1) and 5 -hydroxy -7,3',5' -trimethoxy -4' -(3,3 -dimethylallyloxy) flavone (2) were obtained, and the known flavones 5,6,7,3',4',5' -hexamethoxyflavone (3)⁴, 5 -hydroxy -8,3',4' -trimethoxy -2'',2'' -dimethylpyrano (5",6":6,7) -flavone (4)⁵ and 5 -hydroxy -8,3',4',5' -tetramethoxy -2'' ,2'' -dimethyl-pyrano (5",6":6,7) -flavone (5)⁶. In this paper we describe the isolation and structural elucidation of 1 and 2.

Experimental

General experimental procedures

IR spectra were taken on a Perkin Elmer 281 Spectrometer. ¹H NMR (300 and 500 MHz) and ¹³C NMR (75 and 125 MHz) spectra were recorded on a Varian GEMINI 300 and a Bruker DRX 500, in CDCl₃, with residual solvent peak as int. reference. The MS runs were performed using a Micromass Platform II instrument (Micromass, Wythenshawe, Manchester, UK) equipped with an EI/CI ion source. Electrospray and CAD data were collected in a triple quadrupole Micromass Quatro LC instrument (Micromass, Wythenshawe, Manchester, UK) equipped with a "Z-spray" ion source. Chromatography was performed using silica gel (Merck) for column and preparative TLC. The purity of the compounds was checked by ¹H and ¹³C NMR spectral analysis and TLC plate, revealed with UV (254 nm) and ceric sulphate (0.2 %) in H₂SO₄ (5 %).

Plant material

Leaves of F. gomelleira were collected in Marituba County, State of Pará, Brazil. Voucher specimen n^o 0147615 has been deposited at the Herbarium of the Emílio Goeldi Museum, Department of Botany, Belém, Pará, Brazil.

Extraction and isolation of flavones

The ground dried leaves (3.0 kg) were extracted at room temperature with hexane and CH₂Cl₂, successively. The hexane extract (11.0 g) was chromatographed on silica gel and eluted with a gradient of acetone in hexane to give 20 fractions. Fr. 10 eluted with hexane-acetone (9:1) was purified by repeated column chromatography on silica gel with hexane-CH₂Cl₂-acetone mixtures of increasing polarity, followed by prep. TLC (silica gel, hexane-EtOAc 9:1, 5 runs) to give 2 (3 mg). Fr. 17 eluted with hexane-acetone (8:2) was submitted to repeated CC on silica gel with of hexane-CHCl₃-EtOAc mixtures, followed by prep. TLC (silica gel, developed with CHCl₃, 3 runs) to yield 3 (10 mg).

The dichloromethane extract (29.0 g) was subjected to CC on silica gel and eluted with hexane-CH₂Cl₂-MeOH mixtures of increasing polarity affording 47 fractions. Fr. 22 eluted with hexane-CH₂Cl₂ (6:4) was purified twice by CC on silica gel with mixtures of hexane-CHCl₃-MeOH affording 1 (9 mg). Fr. 28 eluted with hexane-CH₂Cl₂ (1:1) was separated by repeated CC on silica gel with mixtures of hexane-CH₂Cl₂-acetone of increasing polarity affording 5 (16 mg). Fr. 29 eluted with hexane-CH₂Cl₂ (4:6) was purified after repeated CC on silica gel with mixtures of hexane-CHCl₃-acetone, followed by prep. TLC (silica gel, hexane-EtOAc 7:3, 3 runs) to yield 4 (6 mg). The identification of the known compounds 3-5 involved the analysis of ¹H and ¹³C NMR spectral data and comparison with literature values^4-6.

5-hydroxy-7,5'-dimethoxy-3',4' -methylenedioxy-flavone (1). Brown oil. UV l_max/nm (MeOH) (log e): 260 (3.32), 332 (3.09), 380 sh (2.98); UV l_max/nm (MeOH + AlCl₃) (log e): 257 (3.50), 340 (3.06), 380 sh (2.98); UV l_max/nm (MeOH + AlCl₃ + HCl) (log e): 254 (3.60), 341 (3.09), 381 sh (2.99). IR n_max/cm^-1 3405, 1655, 1601, 1491, 1447, 1164, 1045 (film). ¹H and ¹³C NMR see Table 1; EIMS, m/z 342 [M⁺·] (100), 327 [M-CH₃]⁺ (3), 314 [M- CO]⁺· (8), 312 (13), 299 (13), 179 (5), 176 (19), 166 (5).

5-hydroxy-7,3',5'-trimethoxy-4' -(3,3-dimethyl-allyloxy)flavone (2). Brown oil. UV l_max/nm (MeOH) (log e): 245 (3.97), 333 (3.98), 380 sh (3.43); UV l_max/nm (MeOH + AlCl₃) (log e): 277 (4.09), 348 (3.98), 385 sh (3.89); UV l_max/nm (MeOH + AlCl₃ + HCl) (log e): 275 (4.23), 345 (3.97), 380 sh (3.87); IR n_max/cm^-1 3410, 2920, 1648, 1605, 1488, 1447, 1340, 1251, 1116 (film); ¹H and ¹³C NMR see Table 1; ESIMS, m/z 413 [M+H] ⁺ (29), 385 [M+H-CO]⁺ (7), 371 (28), 345 [M+H-C₅H₈]⁺ (100), 329 (29), 301 (30), 298 (9), 171 (14).

Results and Discussion

Compound 1 was isolated as a brown oil and its molecular formula determined as C₁₈H₁₄O₇ from NMR and mass spectral data. The UV spectral data indicated that it was a 5-hydroxyflavone⁷. The chelated hydroxyl hydrogen at d_H 12.70 and the carbonyl group at dC 182.4 were also consistent with a 5-hydroxyflavone^7,8. The aromatic region of the ¹H NMR spectrum (Table 1) displayed five hydrogen resonance signals [d_H 7.06 and 7.09 (d each, J 1.4 Hz), 6.37 and 6.48 (d each, J 2.3 Hz), and 6.54 (s)] which constitute two AB spin systems and an isolated hydrogen. These spectral data were in agreement with a 5,7,3',4',5'-substituted flavone⁷. In addition to a methylenedioxy signal at d_H 6.08, two aromatic methoxyl resonances were observed at d_H 3.88 and 3.99. The fragment peaks at m/z 176 (1a) from a retro-Diels-Alder reaction in the EIMS and m/z 179 (1b) from a retro-Diels-Alder reaction, followed by a rearrangement, were consistent with the ring B substituted with methylenedioxy and methoxy groups at 3',4' and 5' positions, respectively. As a consequence the ring A was substituted with the remaining methoxyl at C-7 and 1 was determined as 5-hydroxy-7,5'- dimethoxy-3',4'-methylenedioxyflavone. The proposed structure of 1 was further confirmed by ¹³C NMR data (Table 1). The correct chemical shifts assignment of all hydrogen (d_H) and carbon (d_C) atoms of 1 was obtained using a combination of HETCOR (¹³C x ¹H-COSY, ¹J(CH); Table 1), HMBC (¹H x ¹³C-COSY, ^2,3J(CH); Figure 1) and NOESY (¹H x ¹H nOe dipolar interaction; Figure 1) experiments. Therefore, for example, the spin-spin interactions of OH-5 (d_H 12.7) with C-5 (d_C 162.2, ²J(CH)), C-6 (d_C 98.2, ³J(CH)) and C-10 (d_C 105.2, ³J(CH)), observed in the HMBC spectrum (Figure 1), have allowed the unequivocal assignment of d_C for C-5, C-6 and C-10. Based on the attribution of C-6 along with a heteronuclear direct one bond coupling between C-6 (d_C 98.2) and H-6 (d_H 6.37) revealed by the HETCOR spectrum, it was possible to establish the unequivocal chemical shift of H-6. As a consequence, the other meta-coupled hydrogen H-8 was attributed at d_H 6.48. The OMe group at position 7 was assigned at d_H 3.88, based on the ¹H x ¹H- NOESY spectrum (Figure 1) which showed the dipolar interactions between CH₃O-7 (d_H 3.88) and both H-6 (d_H 6.37) and H-8 (d_H 6.48), and by cross peak of CH₃O-7 (d_H 3.88) and H-8 (d_H 6.48) with C-7 (d_C 165.6, ^2,3J(CH)), exhibited in the HMBC spectrum. H-2' and H-6' showed doublets at d_H 7.06 and 7.09, respectively. Their assignments were accomplished by the analysis of the NOESY spectrum, which showed a dipolar interaction between CH₃O-5' (d_H 3.99) and H-6' (d_H 7.09). The carbon C-4' is between two oxygenated positions, so it was attributed to the signal at d_C 138.6. The d of the other oxygenated carbon atoms C-3' and C-5' were confirmed by the analysis of the HMBC spectrum, which revealed the cross peaks of OCH₂O (d_H 6.08) with C-3' (d_C 149.6, ³J(CH)) and between H-6' (d_H 7.09) and CH₃O-5' (d_H 3.99) with C-5' (d_C 143.9, ^2,3J(CH)). These data are also in agreement with those reported for the model compounds 5,4'-dihydroxy-7-methoxyflavone⁸ and 5,6,7,8,5'-pentamethoxy-3',4'-methylenedioxy-flavone ⁹.

Compound 2 was isolated as a brown oil and its molecular formula was determined as C₂₃H₂₄O₇ from the NMR and electronspray ionization (ESI) mass spectra. The UV spectral pattern, including applications of shift reagents (see Experimental) and the chelated hydroxyl proton signal at d_H 12.74 indicated that 2 was also a 5-hydroxyflavone⁷. The ¹H NMR (Table 1) of this compound exhibited signals for three aromatic methoxy groups at d_H 3.84 (3H) and 3.94 (6H), one prenyloxy moiety [d_H 4.58 (d, J 7.2 Hz, 2H-1'', 5.56 (t, J 7.2 Hz, H-2'', 1.75 (s, 3H-4'') and 1.68 (s, 3H-5'')] and showed a substitution pattern of ring A identical to compound 1, with two meta-coupled doublets (each J 2.1 Hz) at d_H 6.38 and 6.50, assigned to H-6 and H-8, respectively. The presence of a singlet at d_H 7.07 (2H) assigned to H-2'/6' clearly indicated that ring B was symmetrically substituted with methoxyl groups at 3' and 5' positions. The ¹³C NMR spectrum (Table 1) showed signals for methoxyl groups at d_C 55.8 and 56.4 indicating the absence of methoxyl groups ortho-disubstituted⁸, which implied that the remaining methoxyl was located at C-7 and the prenyloxy group, at C-4'. This was supported by nOe interactions between OMe-7 (d_H 3.84) and both H-6 (d_H 6.38) and H-8 (d_H 6.50), as well as between 2H-1" (d_H 4.58) and OMe-3'/OMe-5' (d_H 3.94) observed in the ¹H x ¹H- NOESY spectrum (Figure 1). The above data led us to formulate the new flavone 2 as 5-hydroxy-7,3',5'-trimethoxy-4'-(3,3-dimethylallyloxy)flavone. The proposed structure 2 was also in agreement with the ¹³C NMR data (Table 1), which were fully assigned using a combination of HETCOR, HMBC and NOESY experiments, as summarized in Table 1 and Figure 1, similarly to what was done with the ¹³C NMR data of 1. These results were also coherent with the data described in the literature for the model compounds 5,4'-dihydroxy-7-methoxyflavone⁸ and 5,6,7,3',5'-pentamethoxy-4'-(3,3-dimethylallyloxy)flavone ².

Acknowledgements

The authors are grateful to the Chemistry Department of UFSCar, São Carlos, Brazil for the EIMS and ESIMS spectra and to the Chemistry Department of UFC, Fortaleza, Brazil for the NOESY and HMBC spectra. We also thank the Emílio Goeldi Museum, Pará, Brazil for the plant identification and CNPq and CAPES Brazilian agencies for the financial support.

Received: March 30, 2000

Published on the web: July 4, 2001

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Publication Dates

• Publication in this collection
  17 Oct 2001
• Date of issue
  Aug 2001

History

• Received
  30 Mar 2000