Luxenchalcone, a new bichalcone and other constituents from Luxemburgia octandra

SHORT REPORT

Luxenchalcone, a new bichalcone and other constituents from Luxemburgia octandra

Mário G. de Carvalho^*, I; Cassia C. Fernandes Alves^I; Kedma G. S. da Silva^I: Marcos N. Eberlin^II; Alceni A. Werle^III

^IDepartamento de Química, ICE, Universidade Federal Rural do Rio de Janeiro, 23851-970 Seropédica - RJ, Brazil

^IIInstituto de Química, Universidade Estadual de Campinas,CP 6154, 13084-971 Campinas - SP, Brazil

^IIIDepartamento de Química, ICEB, Universidade Federal de Ouro Preto, 35400-000 Ouro Preto - MG, Brazil

ABSTRACT

Chromatographic fractionation of leaves and branches extracts from Luxemburgia octandra (Ochnaceae) afforded isoliquiritigenin, 3'-hydroxyisoliquiritigenin, b-sitosterol, stigmasterol, lupeol, betulinic acid, the biflavonoid, 4',5,7-trihydroxyflavone-(3'®O®4'")-5",7"-dihydroxyflavanone and a new bichalcone 4,2',4'-trihydroxychalcone-(3®O®4")-2'",4'"-dihydroxychalcone (luxenchalcone). The structures were established from analysis of IR, MS and NMR spectral data, including 2D-NMR experiments of the natural substances and of the luxenchalcone methyl derivative.

Keywords: Luxemburgia octandra, Ochnaceae, flavonoids, bichalcone, terpenoids

RESUMO

O fracionamento cromatográfico dos extratos de folhas e galhos de Luxemburgia octandra (Ochnaceae) resultou no isolamento de: isoliquiritigenina, 3'-hidroxiisoliquiritigenina, b-sitosterol, estigmasterol, lupeol, ácido betulínico, o biflavonóide, 4',5,7-triidroxiflavona-(3'®O®4'")-5",7" -diidroxiflavanona e uma nova bichalcona, 4,2',4'-triidroxichalcona-(3®O®4")-2'",4'" -diidroxichalcona (luxenchalcona). As estruturas desses compostos foram estabelecidas com base na análise dos dados espectrométricos de IV, EM e RMN incluindo experimentos 2D das substâncias naturais e do derivado metilado da luxenchalcona.

Introduction

Luxemburgia octandra St. Hil (Ochnaceae) is a shrub or small tree distributed throughout the Brazilian Southeastern region. Ochnaceae is a large tropical family including ca. 40 genera and 600 species with the greatest concentration in tropical South America.¹ In the course of our phytochemical and pharmacological investigations of Brazilian plants, we have studied species of both Ouratea and Luxemburgia genera, in which the presence of biflavonoids was observed^2-5 along with their antitumoral activities.^6,7 The biflavonoid 4',5,7-trihydroxyflavone-(3'®O®4'")-5",7"-dihydroxyflavanone was isolated from Luxemburia noilis⁴ and from L. octandra (present work) have shown antitumoral activities.⁶ No previous work has been reported on L. octandra species.

Experimental

General procedures

Melting points are uncorrected. IR spectra were recorded on Perkin-Elmer 1600/1605 FT-IR spectrophotometer using KBr disks or NaCl film. NMR spectra in D₃CCOCD₃, CD₃OD or CDCl₃ were recorded on Bruker (200 MHz for ¹H and 50.3 MHz for ¹³C) and on Jeol JNM-GX-400 (400 MHz for ¹H and 100 MHz for ¹³C) spectrometers using tetramethylsilane (TMS) as internal standard. Electron Ionization Mass Spectra (EIMS) were taken with Gas Chromatography coupled to a Mass Spectrometry (GC/MS) on Varian Saturn 2000 and high resolution electron spray ionization mass spectra (HRESIMS) was obtained with a VG 7070E – HF spectrometer. CC: silica gel (Merck and Aldrich 0.05-0.20 mm); TLC: silica gel H or G (Merck and Aldrich) was used to analyze the fractions collected from column chromatography (CC) with visualization by UV (254 and 366 nm), AlCl₃-EtOH (1%) and exposure to iodine vapor.

Plant material

Luxemburgia octandra St. Hil was collected in Morro de São Sebastião, Ouro Preto, Minas Gerais, Brazil and authenticated by the botanist Jorge L. Silva. A voucher specimen (Nº 26197) is deposited at the OUPR-UFOP Herbarium, Instituto de Ciências Exatas e Biológicas of the Universidade Federal de Ouro Preto-MG, Brazil.

Extraction and isolation

Dried leaves (621g) and branches (1.865 Kg) were powdered and extracted with hexane and ethyl acetate by maceration at room temperature. The extracts were concentrated under vacuum to yield the residues A (6.1 g), and B (10.4 g) from the leaves, C (4.8 g) and D (20.7 g) from the branches, respectively, with hexane and ethyl acetate. The residues B and D were dissolved in H₂O/CH₃OH (1:1) and extracted with hexane/diethyl ether (1:1). These solvents were removed under vacuum and the hexane/diethyl ether fractions (B-1, 0.8 g) and (D-1, 3.5 g) were successively filtered on sephadex LH-20 column using methanol as mobile phase. The collected fractions of B-1 (28 fractions, 50 ml) and D-1 (25 fractions, 50 ml) were analyzed by silica gel TLC. Fractions 15-17 of the B-1 gave biflavonoid 4',5,7-trihydroxy-flavone-(3'®O®4'") -5",7"-dihydroxyflavanone [2, 40 mg, m.p. 220 ºC, [a]_D²⁰ + 1.8 (Me₂CO, c = 0.33)]; fractions 20-24 yielded a mixture of chalcones isoliquiritigenin (3) and 3'-hydroxyisoliquiritigenin (4) (20 mg, gum). The fractions collected from the column of D-1 have impure compound 1 which were crystallized in acetone/ethyl acetate do yield pure compound 1 (yellow crystals, 300 mg, mp 172 ºC). Product 1a (yellow crystals, 50 mg, mp 168 ºC) was obtained by treating a methanol solution of 1 (50 mg) with ethereal diazomethane. The residue C was dissolved in methanol and successively submitted to column chromatography of sephadex LH-20 using methanol as mobile phase. Fraction C-10/14 yielded steroids mixture, sitosterol and stigmasterol (50 mg); fractions C-18/19 and C-22/27 gave the triterpene lupeol (20 mg, mp 202 ºC) and betulinic acid (30 mg, mp 295 ºC), respectively.

4,2',4'-Trihydroxychalcone-(3®O®4")-2'",4'"-dihydroxychalcone (luxenchalcone), (1)

IR (KBr) n_max/cm^-1: 3497, 1630, 1510, 1432, 1289, 1225, 974, 837, 802. ¹H NMR (D₃CCOCD₃, 200 MHz) and ¹³C NMR (D₃CCOCD₃, 50.3 MHz) (Table 1). HRESIMS/MS: m/z (rel. int.) 510.0584 [M^+•,15 (calcd 510.131467 for C₃₀H₂₂O₈)], 399.0673 (10), 373.0878 (100), 355.0829 (23), 135.0125 (53).

4,4'-Dimethoxy-2'-hydroxychalcone-(3®O®4")-2'"-hydroxy-4'"-methoxychalcone (luxenchalconetrimethylether), (1a)

IR (KBr) n_max/cm^-1: 3410, 2923, 2851, 1638, 1562, 1508, 1461, 1361, 1270, 1227, 1124, 972, 842. ¹H NMR (CDCl₃, 400 MHz) and ¹³C NMR (CDCl₃ 100 MHz) (Table 1). EIMS m/z (rel. int.) 552 (M⁺ , 33% ,C₃₃H₂₈O₈), 401 (65), 151 (100).

Results and Discussion

The known natural compounds b-sitosterol, stigmasterol,^10,11 lupeol,¹² betulinic acid,¹³ isoliquiritigenin (3),⁸ 3-hydroxy-isoliquiritigenin (4)⁹ and 4',5,7-trihydroxy-flavone-(3'®O®4'")-5",7"-dihydroxy flavanone (2)^4,14 were identified mainly by ¹H and ¹³C-NMR spectral analyses besides comparison with literature data.^4,5,8-14

Compound 1 gave a positive test with AlCl₃-EtOH reagent, which confirmed the presence of a flavonoid. The IR spectrum showed bands at 3497 (n_O-H), 1630 (n_C=O) and 1510, 1432 cm^-1 attributed to aromatic ring. The 1D and 2D ¹H NMR spectra showed signals of three ABC systems, d 6.34 (d, 1H), 6.36 (d, 1H), 6.46 (dd, 2H), 8.10 (d, 2H) and 7.09 (d, 1H), 7.57 (dd, 1H) and 7.69 (d, 1H) and signals of AA'BB' at d 7.00 (d, 2H) and 7.84 (d, 2H) of four aromatic rings. The additional signal at d 13.58 (s, 2H) of two chelated hydroxyls and a multiplet at d 7.83 and 7.85 for hydrogens connected at a and b carbons [(d_CH 117.2 and 143.2 as observed in the ¹H,¹³C-COSY (¹J_CH)] spectra, besides the d_C=O at 192.4, allowed to propose the chalcone structure (Table 1). These data and ¹³C NMR (PND and DEPT) and ¹H, ¹³C-COSY (ⁿJ_CH n = 1,2,3) spectra analysis were in agreement with two chalcones reported in the literature, isoliquiritigenin (3)⁸ and 3-hydroxy-isoliquiritigenin (4).⁹ Treatment of 1 with diazomethane yielded 1a with three methoxy groups, besides two chelated hydroxy groups (Table 1). These information allowed us to propose the structure of a new bichalcone 4,2',4',2'",4'"-pentahydroxy-3®O®4"-bichalcone (1, named as luxenchalcone) (Table 1). The C-3-O-C-4" connection between the chalcone moieties was confirmed by a NOESY and NOEDIFF experiment of the derivative 1a. Irradiation at the methoxy groups showed NOE at d 7.06 (d, 8.4 Hz, H-5), and at signals near d 6.40 (H-3', H-5', H-3'", H-5'"). These experiments did not show NOE at d 7.43 (d, H-2) and 6.97 (d, H-3", H-5"). This result indicates that the positions 3 and 4" are not substituted with OCH₃ groups. The 2D experiments [¹H,¹H-COSY, ¹H,¹³C-COSY (HETCOR and COLOC of 1 and HMQC and HMBC of 1a)] were used to confirm the ¹H and ¹³C chemical shifts of 1 and 1a (Table 1). The cross peaks observed in the HMBC spectra of 1a show long-range couplings of C-4 (d_C 153.8) with OCH₃ (d 3.88), with H-2 and with H-6 (³J_CH) and also of C-3 (d_C 144.0) with H-2 (²J_CH) and H-5 (³J_CH) which were used to confirm the C-3-O-C-4" connection. The peaks at m/z 552 (M^+•, 33%, C₃₃H₂₈O₈) in the LREIMS of 1a and the m/z 510.0584 (M^+•, 15%; C₃₀H₂₂O₈ requires 510.13147) in the HRESIMS of 1 were also used to confirm the proposed structure of this new bichalcone.

Acknowledgements

The authors are grateful to CNPq, CAPES and FAPERJ for scholarships and financial support, as well as to J. Schripsema and R. Braz-Filho, LCQUI-CCT-UENF, for the 400 MHz NMR spectra of 1a.

Received: January 6, 2003

Published on the web: October 28, 2003

FAPESP helped in meeting the publication costs of this article.

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Publication Dates

• Publication in this collection
  25 May 2004
• Date of issue
  Feb 2004

History

• Accepted
  28 Oct 2003
• Received
  06 Jan 2003