A new isoflavone isolated from Harpalyce brasiliana

Article

A New Isoflavone Isolated from Harpalyce brasiliana^*

Graça Lúcia da Silva ^a , Maria Iracema Lacerda Machado ^a ,Francisco José de Abreu Matos ^a , and Raimundo Braz-Filho ^b**

^aDepartamento de Química Orgânica e Inorgânica, Laboratório de Produtos Naturais, Universidade Federal do Ceará, C.P. 12200, 60.021-970 Fortaleza - Ceará, Brazil

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

Uma nova isoflavona denominada harpalicina e quercetina foram isoladas das folhas e 3-hidroxi-4-isopentenil-8,9-metilenodioxipterocarpano e ácido betulínico das raizes de Harpalyce brasiliana. As estruturas foram elucidadas usando métodos espectrométricos, inclusive RMN bidimensional (2D) da nova isoflavona.

A new isoflavone named harpalycine and quercetin were isolated from the leaves and 3-hydroxy-4-isopentenyl-8,9-methyle-nedioxypterocarpan and betulinic acid from the roots of Harpalyce brasiliana. The structures were elucidated using spectroscopic methods, including 2D NMR techniques of the new isoflavone.

Keyword: Harpalyce brasiliana, Leguminosae, flavonoids, triterpene

Introduction

Harpalyce brasiliana Benth. (Leguminosae-Papilionoideae) is a Northeastern Brazilian shrub, called "raiz-de-cobra" (snake root) and used by people for treating snake bites¹. 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, bredemeyeroside³.

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^4-6.

Results and Discussion

Chromatographic separation of the ethanol extract from the leaves of Harpalyce brasiliana led to the isolation of the new isoflavone harpalycin (3), as well as the known flavonol quercetin (4). From the roots, 3-hydroxy-4-isopentenyl-8,9-methylenedioxypterocarpan (5) and pentacyclic triterpenoid betulinic acid (6) were isolated.

The known natural products quercetin (4, 5,7,3’,4’-tetrahydroxyflavonol) and betulinic acid [6, 3b-hydroxy-20(29)-lupen-28-oic acid] were identified mainly by their ¹H and ¹³C-NMR spectra and comparison with literature data^7,8. 3-Hydroxy-4-isopentenyl-8,9-methylenedioxypterocarpan (5) has been recently reported^5,6.

Comparative analysis of the hydrogen broad band decoupled (HBBD) and distortionless enhancement by polarization transfer (DEPT) ¹³C-NMR spectra⁹ of 3, in combination with the ¹H-NMR (one- and two-dimensional ¹Hx¹H-COSY), IR [n 3420 (OH), 1660 (conjugated carbonyl), 1620 (conjugated double bond), 1590 and 1500 cm^-1 (aromatic ring)] and mass {m/z 382 ([M]⁺, 70 %), 364 ([M - H₂O]⁺, 12 %), 349 ([M - H₂O - Me^.]⁺, 39 %), 311 (3c, [M - C₄H₇O]⁺, 100 %), 310 (3d, [M - C₄H₈O]⁺, 45 %) and 146 (3e, 18 %)} spectra allowed the deduction of a molecular formula C₂₁H₁₈O₇, containing eleven quaternary carbons {ten sp²: one carbonyl (d_C 180.08, C-4), five bound to oxygen atoms [d_C 159.27 (C-7), 159.09 (C-5), 155.19 (C-9), 147.70 (C-3’ and C-4’)], four non-oxygenated [d_C 121.71 (C-1’) 124.47 (C-3), 104.22 (C-6 and C-10)] and one sp³ oxygenated [d_C 79.03 (C-2’’], six methine [five sp²: d_C 154.70 (CH-2), 122.57 (CH-6’), 109.42 (CH-2’), 108.19 (CH-5’) and 94.22 (CH-8); one sp³ bound to oxygen: d_C 66.80 (CH-3’’)], two methylene [d_C 101.16 (3’,4’-OCH₂O) and 25.02 (CH₂-4’’)] and two methyl groups [d_C 25.25 (CH₃-6’’) and 21.07 (CH₃-5’’)]: (C)₁₀ (O)₄(C=O) (CH)₆(OCH₂O)(CH₂)(CH₃)₂ = C₂₁H₁₆O₇. The two remaining hydrogens (C₂₁H₁₈O₇, m/z 382 [M]⁺, 70 %) were attributed to two hydroxy groups: (C)₁₀(O)₂(C=O)(CH)₆ (OCH₂O)(CH₂)(CH₃)(OH)₂=C₂₁H₁₈O₇. The presence of the two hydroxy groups was confirmed by the singlet signals at d_H 2.04 (AcO-3’’) and 2.43 (AcO-5) observed in the ¹H-NMR spectrum and d_C 170.36 and 20.97 (AcO-3’’) and 169.12 and 21.08 (AcO-5), in the ¹³C-NMR spectrum of the diacetyl derivative 3b (Table 1). One chelatogenic hydroxyl function was revealed by signals at d_H 13.20 and 13.13 (HO-5) in the ¹H-NMR spectra of 3 and monoacetyl derivative 3a, respectively. These data and the signals at d_H 8.12 (H-2) and d_C 154.70 (CH-2) were used to classify this natural product as an isoflavone containing one methylenedioxy and one monohydroxylated isoprenoid moiety (Me₂C-CHOH-CH₂-) involved in a 3,4-dihydro-3-hydroxy-2,2-dimethylpyran ring, along with the chelatogenic hydroxyl group at C-5 (d_H 13.20).

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 d_H 3.69 (dd, J = 6.8 and 5.6 Hz) and 5.12 (dd, J = 5.1 and 4.7 Hz), observed in the ¹H-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 d_C 69.76 (CH-3’’) and signals at d_H 1.37 (3H-5’’, ³J_CH) and d_H 1.32 (3H-6’’, ³J_CH), observed in the 2D ¹³Cx¹H-COSY ⁿJ_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 (d_H 13.13) and quaternary carbon atoms C-5 (d_C 160.11, ²J_CH), C-6 (d_C 102.72, ³J_CH) and C-10 (d_C 105.39, ³J_CH), as summarized in Table 2. Additional long range heteronuclear correlations observed in the 2D ¹³Cx¹H-COSY-ⁿJ_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 ¹³Cx¹H-COSY-¹J_CH spectrum of 3a (Table 1). Thus, the complete assignment of the chemical shifts of hydrogen and carbon atoms of 3a in the ¹H and ¹³C-NMR spectra was accomplished by 2D ¹Hx¹H-COSY and ¹³Cx¹H-COSY ⁿJ_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 signals⁹.

Thus, the structure of the new isoflavone, named harpalycine, isolated from Harpalyce brasiliana was established as 3*,4-dihydro-3,5-dihydroxy-7-(3,4-methylenedioxyphenyl)-2,2-dimethyl-2H,6H-benzo

[1,2-b:5,4-b’] dipyran-6-one (3, 3’’*,5-dihydroxy-2’’,2’’-dimethyl-3’,4’-methylenedioxy

-6,7:6’’,5’’-pyranoisoflavone). Relatively few dihydrohydroxypyranoisoflavones have been described as natural products^11,12.

Experimental

General experimental procedures

Mps are uncorr. IR spectra were recorded on a Perkin Elmer 1320 or Nicolet 5ZDXFT-IR, in KBr. ¹H [400 (3) and 270 (3a and 3b) MHz] and ¹³C [50 (3) and 67.5 (3a and 3b) MHz] NMR spectra were recorded on a Bruker AC-200 (¹H: 200 MHz; ¹³C: 50 MHz) and WP-270 (¹H: 270 MHz; ¹³C: 67.5 MHz) or Varian UN-400 (¹H: 400 MHz; ¹³C: 100 MHz) spectrometers, in pyridine-d₅ (3, ¹H-NMR), DMSO-d₆ (3, ¹³C-NMR) or CDCl₃ (3a and 3b). EIMS (70 eV) spectra were obtained on a HP-5971 GC/MS instrument.

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, CHCl₃, EtOAc, CHCl₃-MeOH (1:1), MeOH and EtOH. The residue (55 g) of the fraction eluted with CHCl₃-MeOH (1:1) was suspended in EtOH-H₂O soln and extracted with hexane; the residue (23 g) thus obtained was chromatographed on a silica gel column and eluted with hexane, CHCl₃, EtOAc and MeOH; the fraction eluted with CHCl₃ (2 g) was recrystallized from MeOH to afford 3 (286 mg); the fraction eluted with EtOAc (10 g) furnished quercetin (4, 50 mg) after several runs on a silica gel column.

Extraction and isolation of constituents from roots

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

Harpalycin (3)

Mp 208 - 211° (MeOH). IR n_max (cm^-1, KBr): 3420, 1620, 1660, 1590, 1500, 1190, 820. EIMS m/z (rel. int.): 382 ([M]⁺, 70), 364 ([M - H₂O]^+., 12), 349 ([M - H₂O - Me^.]⁺, 39), 311 (3c, 100), 310 (3d, 45), 146 (3e, 18), 145 ([3e - H^.], 13). H¹ (400 MHz, C₅D₅N) and ¹³C (50 MHz, DMSO-d₆) NMR: Table 1.

Acetylation of harpalycin (3)

Treatment of harpalycin (3, 100 mg) with Ac₂O (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 CHCl₃ as eluents.

3’’-O-Acetylharpalycin (3a)

Mp 212-213°. IR n_max (cm^-1, KBr): 1730, 1650, 1620, 1590, 1500, 1190 e 800. EIMS m/z (rel. int.): 424([M]⁺, 6), 364 ([M - AcOH]⁺, 8), 349 ([M - AcOH - Me^.]⁺, 54), 146 (3e, 44); 145 ([3e - H^.]⁺, 46). ¹H (270 MHz, CDCl₃) and ¹³C (67.5 MHz, CDCl₃) NMR: Table 1; ¹³C-¹H COSY ¹J_CH and ¹³C-¹H COSY ⁿJ_CH (n = 2 and 3, COLOC) NMR (¹H: 200 MHz; ¹³C: 50 MHz): Table 2.

3’’,5-Di-O-Acetylharpalycin (3b)

Mp 203-204°. IR n_max (cm^-1, KBr): 1730, 1620, 1590, 1500, 1190, 800. EIMS m/z (rel. int.): 466 ([M]⁺, 6), 406 ([M - AcOH]⁺, 2), 364 ([M - AcOH - CH₂=C=O]⁺, 8), 349 ([M - AcOH - CH₂=C=O - Me^.]⁺, 100), 146 (3e, 44). ¹H (270 MHz, CDCl₃) and ¹³C (67.5 MHz, CDCl₃) NMR: Table 1.

Acknowledgments

The authors are grateful to Professor Afrânio G. Fernandes (Botanist, Departamento de Biologia, Universidade Federal do Ceará) for plant identification; to CAPES, PADCT/FINEP and CNPq for financial support and CNPq for a research fellowship (R.B-F). We thank Dr. Victor Rumjanek for reading the manuscript.

Received: May 31, 1999

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^*

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.

Publication Dates

• Publication in this collection
  01 June 2001
• Date of issue
  Dec 1999

History

• Received
  31 Mar 1999