A New Isoflavone Isolated from Harpalyce brasiliana *

Harpalyce brasiliana Benth. (Leguminosae-Papilionoideae) is a Northeastern Brazilian shrub, called “raizde-cobra” (snake root) and used by people for treating snake bites. Two prenylated pterocarpans, cabenegrins A-I (1) and A-II (2), potent antidotes against snake venom, were isolated and identified from a locally well known anti-snake bite medicine named “Específico Pessoa”, manufactured and sold in the north and northeast of Brazil and available to plantation workers as an oral antidote. The plant, commonly called “cabeça de negro”, which furnishes the extract used in the preparation of this remedy has not been identified so far, being kept secret by the manufacturers. There are about ten plants with the name “cabeça de negro” in South America. Two plants reputed as anti-snake bite medicines occur in the Ibiapaba region in Northeast Brazil: Bredemeyera floribunda Willd (Polygalaceae), called “pacari”, and Harpalyce brasiliana (LeguminosaePapilionoideae). The first contains as its active principle one saponin, bredemeyeroside. In this paper we report the isolation and characterization of the new isoflavone harpalycin (3) and the known flavonol quercetin (4), prenylated pterocarpan (5) and triterpene betulinic acid (6) from leaves and roots of a specimen of Harpalyce brasiliana. There are three previous chemical reports about this plant.


Introduction
Harpalyce brasiliana Benth.(Leguminosae-Papilionoideae) is a Northeastern Brazilian shrub, called "raizde-cobra" (snake root) and used by people for treating snake bites 1 .Two prenylated pterocarpans, cabenegrins A-I (1) and A-II (2) 2 , potent antidotes against snake venom, were isolated and identified from a locally well known anti-snake bite medicine named "Específico Pessoa", manufactured and sold in the north and northeast of Brazil and available to plantation workers as an oral antidote.The plant, commonly called "cabeça de negro", which furnishes the extract used in the preparation of this remedy has not been identified so far, being kept secret by the manufacturers.There are about ten plants with the name "cabeça de negro" in South America.Two plants reputed as anti-snake bite medicines occur in the Ibiapaba region in Northeast Brazil: Bredemeyera floribunda Willd (Polygalaceae), called "pacari", and Harpalyce brasiliana (Leguminosae-Papilionoideae).The first contains as its active principle one saponin, bredemeyeroside 3 .
In this paper we report the isolation and characterization of the new isoflavone harpalycin (3) and the known flavonol quercetin (4), prenylated pterocarpan (5) and triterpene betulinic acid (6) from leaves and roots of a specimen of Harpalyce brasiliana.There are three previous chemical reports about this plant [4][5][6] .
The location of hydroxy group at carbon atom CH-3'' of the 3,4-dihydro-3-hydroxy-2,2-dimethylpyran moiety was deduced by signals at δH 3.69 (dd, J = 6.8 and 5.6 Hz) and 5.12 (dd, J = 5.1 and 4.7 Hz), observed in the 1 H-NMR spectra of 3 and 3a, respectively (Table 1), whose attribution was confirmed by a long range heteronuclear correlation (spin-spin interaction) of the signal at δC 69.76 (CH-3'') and signals at δH 1.37 (3H-5'', 3 J CH ) and δH 1.32 (3H-6'', 3 J CH ), observed in the 2D 13 Cx 1 H-COSY n J CH (n = 2 and 3, COLOC) 10 spectrum of the monoacetyl derivative 3a (Table 2).This spectrum also showed correlation of the signals corresponding to the hydrogen of the hydroxy group at C-5 (δ H 13.13) and quaternary carbon atoms C-5 (δ C 160.11, 2 J CH ), C-6 (δ C 102.72, 3 J CH ) and C-10 (δ C 105.39, 3 J CH ), as summarized in Table 2.Additional long range heteronuclear correlations observed in the 2D 13 Cx 1 H-COSY-n J CH (n = 2 and 3, COLOC) spectrum of 3a are summarized in Table 2, along with the heteronuclear direct one bond coupling revealed by the 2D 13 Cx 1 H-COSY-1 J CH spectrum of 3a (Table 1).Thus, the complete assignment of the chemical shifts of hydrogen and carbon atoms of 3a in the 1 H and 13 C-NMR spectra was accomplished by 2D 1 Hx 1 H-COSY and 13 Cx 1 H-COSY n J CH (n = 2 and 3, COLOC), which confirmed the proposed structure 3 (Table 1 and 2).These assignments were facilitated by application of the usual shift parameters and the observed multiplicities of signals 9 .

General experimental procedures
Mps are uncorr.IR spectra were recorded on a Perkin Elmer 1320 or Nicolet 5ZDXFT-IR, in KBr.

Plant material
Harpalyce brasiliana Benth.leaves and roots were collected in Guaraciaba do Norte, Ibiapaba mountains, Ceará State, Brazil and identified by Professor Afrânio Gomes Fernandes (Universidade Federal do Ceará, Fortaleza).A voucher specimen (n o 14841) is deposited at the Herbário Prisco Bezerra of the Departamento de Biologia of the Universidade Federal do Ceará.

Extraction and isolation of constituents from leaves
Dried and powdered leaves (6 kg) were extracted with EtOH at room temp and the solvent removed under vacuum to yield 736 g of residue.This residue was chromatographed on a silica gel column and successively eluted with hexane, CHCl 3 , EtOAc, CHCl 3 -MeOH (1:1), MeOH and EtOH.The residue (55 g) of the fraction eluted with CHCl 3 -MeOH (1:1) was suspended in EtOH-H 2 O soln and extracted with hexane; the residue (23 g) thus obtained was chromatographed on a silica gel column and eluted with hexane, CHCl 3 , EtOAc and MeOH; the fraction eluted with CHCl 3 (2 g) was recrystallized from MeOH to afford 3 (286  1) NMR spectra were also used for these assignments.Chemical shifts and coupling constants (J) of hydrogen atoms obtained from 1D 1 H-NMR spectrum.

Extraction and isolation of constituents from roots
The natural products 5 and betulinic acid (6) were isolated from roots as described in Ref. 6.

Acetylation of harpalycin (3)
Treatment of harpalycin (3, 100 mg) with Ac 2 O (4 mL) in the presence of pyridine (1 mL), and usual work-up, produced a mixture of 3a (monoacetyl derivative) and 3b (diacetyl derivative), which were purified on a silica gel column using hexane and hexane containing increasing amount of CHCl 3 as eluents.
This work is part of the M.Sc.Thesis of Graça Lúcia da Silva, presented at the Departamento de Química Orgânica, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil, 1995.