Diploflavone , a New Flavonoid from Diplotropis ferruginea Benth . ( Fabaceae )

Jackson Roberto G. S. Almeida, José Maria Barbosa-Filho*, Analúcia G. S. Cabral , Maria de Fátima Agra, Emidio V. Leitão da-Cunha, Marcelo S. da Silva, Silene C. do Nascimento and Raimundo Braz-Filho Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, CP 5009, 58051-970 João Pessoa PB, Brazil Universidade Federal do Vale do São Francisco, CP 252, 56306-410 Petrolina PE, Brazil


Introduction
The Fabaceae have a cosmopolitan distribution, consisting of ca 700 genera and more than 17000 species. 1 The genus, Diplotropis consists of approximately 22 species, including, Diplotropis ferruginea Benth.Investigations of only two species have been reported in the literature: the isolation of quinolizidine alkaloids from Diplotropis martiusii, 2 and flavonoids from Diplotropis purpurea. 3iplotropis ferruginea is a tree native to Northeastern Brazil, where it is popularly known as "sucupira".It is used in folk medicine for the treatment of rheumatism, arthritis and diabetes. 4Recently, a chemical investigation of this species resulted in the isolation of lupeol, ethyl 2-hydroxy-4-methoxy-6-propyl benzoate 5 and of the flavonoid 3,4,5,8-tetramethoxy-6,7,2",3"-furanoflavan. 6pasmolytic activity was reported for the crude EtOH extract of this plant. 7his paper describes the isolation of two more flavonoids, whose structures were established by spectroscopic techniques, mainly EIMS and 1D and 2D NMR.

General experimental procedures
Melting points were determined on a REICHERT, model R3279 "Kofler" apparatus, and are uncorrected.IR spectra were obtained in KBr on a BOMEM model MB 100 spectrophotometer. 1 H and 13 C NMR spectra were run on a Jeol Eclipse+ 400 spectrometer operating at 400 MHz for 1 H and 100 MHz for 13 C, using CDCl 3 as solvent (approximately 10 mg of sample were dissolved in 0.5 mL of solvent and transferred into a 5 mm NMR tube) and solvent signals were used as internal reference for the chemical shifts δ H 7.26 (CHCl 3 ) and δ C 77.00 (CDCl 3 ).The one-dimensional (1D) 1 H and 13 C NMR spectra were acquired under standard conditions (5 mm multinuclear probe).The two-dimensional (2D) experiments were acquired and processed with the Delta software provided by Jeol.Standard pulse sequences were used for all experiments. 1H-1 H-COSY spectra were obtained with X-points 512/Y-points 256, X-resolution 11.7 Hz/Yresolution 23.4 Hz, X-acquisition time 85.4 ms/Y-acquisition time 42.7 ms, pulse 90 o , relaxation delay 1.5 s, zerofill: 4. For homonuclear 2D 1 H-1 H-NOESY experiments were used mixing time 0.5 s, X-points 512/Y-points 256, X-resolution 11. 7Hz/Y-resolution 23.4 Hz, X-acquisition time 85.4 ms/ Y-acquisition time 42.7 ms, pulse 90 o relaxation delay 1.5 s, zerofill: 4. Two-dimensional inverse hydrogen detected heteronuclear shift correlation 1 H-13 C-HMQC-1 J CH spectra were obtained with 1 J CH = 140 Hz, X-points 1024/Y-points 128, X-resolution 5.86 Hz/Y-resolution 196 Hz, X-pulse 90 o / Y-pulse 90 o , X-acquisition time 0.17 s/Y-acquisition time 5.09 ms, pulse 90 o , relaxation delay 2.0 s, gradient 1/3 1 ms square, zerofill: 4. Two-dimensional inverse hydrogen detected heteronuclear long-range correlation 1 H-13 C-HMBC-n J CH (n=2 and 3) experiments were carried out by using n J CH = J constant 140 Hz/J long range 8 Hz, X-points 1024/Y-points 128, X-resolution 5.86 Hz/Y-resolution 196 Hz, X-pulse 90 o /Y-pulse 90 o , X-acquisition time 0.17 s/Yacquisition time 5.09 ms, pulse 90 o , relaxation delay 2.0 s, gradient 1/3 1 ms square, zerofill: 4. EIMS were measured at 70 eV on a GC/MS System Shimadzu QP-5050.

Plant material
The stem bark of Diplotropis ferruginea was collected in the municipality of Caraúbas, State of Rio Grande do Norte, Northeastern Brazil in May 2002.Botanic material was identified by Prof. Maria de Fátima Agra, of the Laboratório de Tecnologia Farmacêutica.A voucher specimen (AGRA & D. ALMEIDA 5559) is deposited at the Herbario Prof. Lauro Pires Xavier (JPB), of the Universidade Federal da Paraíba.

Extraction and isolation
The dried and powdered stem bark of D. ferruginea (3 kg) was exhaustively extracted with 95% EtOH at room temperature.The extract was concentrated under vacuum yielding 95 g of the crude product.This was suspended in a MeOH:H 2 O (3:7 v/v) mixture and partitioned with hexane, CHCl 3 and EtOAc.The hexane fraction was then subjected to silica gel column chromatography and eluted with hexane, CHCl 3 and MeOH in an increasing polarity gradient to give 152 fractions.The fractions were monitored by TLC and classified into 25 groups.Fraction 97-102 was purified by preparative TLC over silica gel using CHCl 3 :MeOH (9:1) to afford flavonoid 1 (61 mg) and the fraction 89-96 was purified in the same way using hexane:EtOAc (2:1) to afford flavonoid 2 (123 mg).

Biological assay
The cytotoxic activity assays were based on the methylazoetetrazolium (MTT) method or the 3-(4,5dimethylazol-2-yl)-3,5-diphenyltetrazolium bromide method. 8For the evaluation of cytotoxity the cellular strain HEp2 (larynx carcinoma) NCIH-292 (lung carcinoma) KB (mouth carcinoma) 9 with proven viability were used.The cells were grown in MEM-Minimal Essential Medium 10 with 10% bovine fetal serum containing 1% antibiotics solution (penicillin 1000 UI mL -1 + streptomycin 250 mg mL -1 ) and 1% glutamine (200 μM).A cellular suspension of 5 10 4 cells mL -1 was used and distributed in plates of 96 wells.The test samples of 0.15 mL were added into each well.The plates were incubated for 72 h at 37 o C in a humid atmosphere enriched with 5% CO 2 .After incubation 15 mL MTT in phosphate buffered saline (BPS) solution at (5 mg mL -1 ) was added into each well.After 2h the culture medium was removed and 100 μL of DMSO were added in each well for quantitation of blue formazan.The readings were performed with the aid of a Multskan ELX 800 cell reader (Bio-Tec Instruments -USA) at 540 nm.

Results and Discussion
Flavonoid 1 was obtained as a colorless amorphous solid.Its molecular formula was deduced as C 26 H 26 O 5 (14 degrees of unsaturation), supported by the occurrence of the molecular ion at m/z 418 in the MS, in combination with1 H and 13 C-APT-NMR spectral data.The IR spectrum showed absorptions at 1620 cm -1 , attributed to an α-β unsaturated carbonyl group; 3062 cm -1 attributed to unsaturated C-H and absorptions in the region 1379-1404 cm -1 , suggesting the presence of a gem-dimethyl group. 1 H NMR of 1 showed signals at δ H 8.07 (2H, br, d J = 7.7 Hz) and 7.56-7.46(3H, m) which indicates the possibility of a mono-substituted ring B in a flavonoid.The presence of a 2,2-dimethylchromene moiety was indicated by the characteristic signals of its two vinyl hydrogens forming an AB system 11 at δ H 5.74 (1H, d, J = 9.9 Hz) and 6.87 (1H, d, J = 9.9 Hz) and a signal at δ H 1.54 (6H, s) attributed to the two methyl groups.A signal at δ H 1.79 (6H, s) was also observed and signals at δ H 4.68 (1H, d, J = 6.2 Hz) and 5.53 (1H, t, J = 6.2 Hz), suggesting the presence of a prenyl group in the molecule.This suggestion is confirmed by the 13 C-APT NMR spectra which shows signals at δ C 18.25 and 25.72, for 2 methyl carbons and a methylene carbon at δ C 66.29.The chemical shift of the methylene carbon in the 13 C NMR indicates that the prenyl group is bound to an oxygen atom.The HMBC experiment showed the location of the O-prenyl group at C-6, due to the3 J CH correlation between the signal at δ H 4.68 (prenyl's methylene hydrogens) with the signal at δ C 146.06 (C-6).The analysis of all the spectral data for 1 led to the elucidation of its structure as 3-methoxy-6-O-prenyl-6",6"-dimethylchromene-(7,8,2",3")-flavone.This substance is described here for the first time and was given the trivial name diploflavone.
Flavonoid 2 was isolated as a colorless amorphous solid.Its molecular formula deduced as C 22 H 20 O 5 (13 degrees of unsaturation), was confirmed by the molecular ion at m/z 364 in the MS in combination with 1 H-NMR (1D and 2D 1 H-1 H-COSY) and 13 C-APT-NMR spectral data.IR and 1 H and 13 C-NMR spectra showed the similarity with substance 1.The only difference between the two substances was the absence of the prenyl moiety in 2, having a methoxy in the same position.The presence of the methoxy group was indicated by the signal at δ H 3.97 (3H, s).The substance was thus characterized as the flavonoid 3,6-dimethoxy-6",6"-dimethylchromene-(7,8,2",3")-flavone (2), previously isolated from Bowdichia virgilioides and the NMR data are in accordance with the literature. 12he 2D experiments HMQC and HMBC were used to confirm the 1 H and 13 C chemical shifts of 1 (Table 1) and 2. 12

Table 1 .
1H (400 MHz) and 13 C (100 MHz) NMR data for 1 including results obtained by heteronuclear 2D shift-correlated HMQC and HMBC spectra, in CDCl 3 as solvent and TMS as internal reference.Chemical shifts in δ (ppm) and coupling constants (J, in parenthesis) in Hz* * Homonuclear 1 H-1 H-COSY spectra were also used for these assignments.Chemical shifts of hydrogen atoms obtained from 1D