Chemical Constituents from Ouratea floribunda : Complete 1 H and 13 C NMR Assignments of Atranorin and its New Acetyl Derivative

O fracionamento cromatográfico do extrato hexânico da madeira de Ouratea floribunda (Ochnaceae) forneceu, além dos triterpenos friedelina (1), friedelanol (2) e lupeol (3), o depsídeo atranorina (4). As estruturas das substâncias isoladas foram determinadas através da análise dos dados espectroscópicos de RMN 1H e 13C, massas e comparação com dados da literatura. A atribuição inequívoca dos deslocamentos químicos dos átomos de carbono e hidrogênio do depsídeo 4 e do seu novo derivado monoacetilado 4a foi realizada através de técnicas unie bidimensionais de RMN (1H-1H COSY e NOESY, 1H-13C HMBC e HMQC) e está sendo descrita pela primeira vez na literatura.


Introduction
The genus Ouratea (Ochnaceae) comprises ca 300 tropical species occurring mainly in South America 1 .Some species of this genus have been shown to possess antiviral (Ouratea lucens, from Panama) 2 , antimicrobial (O.parviflora, from Brazil) 3 as well as pain relief (O.reticulata, from Guinea) activities 4 .The species O. spectabilis, from Brazil, has been reported to be used in folk medicine for the treatment of rheumatic and gastric distress.Recently, the inhibition of bovine lens aldose reductase by a biflavone isolated from this species has been described 5 .
As part of our chemical and pharmacological studies of Brazilian plants we reported the investigation of some Ouratea species [8][9][10][11] .In those studies, we also detected the inhibition of murine tumour growth and an antiproliferative effects and activation of apoptosis on Ehrlich tumour cells by biflavones isolated from two species belonging to this genus 12 .
In this first study of O. floribunda we report the presence of known triterpenes (1 -3) and describe the spectroscopic data of the depside 4 and of its new monoacetyl derivative 4a.The structure elucidation of the isolated compounds was performed by spectrometric analysis involving comparison with literature data.The unambiguous assignments of 1 H and 13 C NMR data of atranorin 4 and of its monoacetyl derivative 4a are reported for the first time and involved 1 H-1 H homonuclear (COSY and NOESY) and 1 H- 13 C heteronuclear (HMQC and HMBC) NMR experiments.
The molecular formula of were used to propose two aromatic rings sustaining five substituents (vide infra) and one hydrogen atom [δ H 6.50 (s) and 6.39 (s)] each.
The acetylation of 4 at 60 o C yielded the hydrolysis product, methyl 2-hydroxy-4-hydroxy-3,6-dimethylbenzoate (4b).The same acetylation carried out at room temperature gave the new monoacetate 4a, whose structure was confirmed by complete 1 H and 13 C NMR spectra assignment (Table 1).As was expected, the chemical shift of H-6' was shifted from δ H 6.50 in 4 to δ H 6.91 in 4a, with its location at ortho or at para positions and, in accordance with the presence of H-6' para in relation to the 3'-hydroxyl group in 4 19 .This is the first report for 13 C NMR data assignments for the depside 4 Scheme 1. Proposed fragmentation mechanisms of 4 (only peaks classified as principal).
Depsides are aromatic products of polyketide origin which are especially well represented in lichens, the most characteristic being formed by the condensation of two or three simple orcinol or β-orcinol-type phenolic moieties linked by an ester bond.A large number of structurally related metabolites have been detected in several lichens.Although recently a tridepside fenviorin was isolated from the plant Frullania nisqualensis 20 .Atranorin is a para-β-orcinol depside bearing CO 2 Me, CHO and OH groups, frequently found in lichens, it seems that the wood of O. floribunda used in this study was contaminated.Among the aromatic lichen compounds, atranorin is the most frequent allergenic agent responsible for airborne contact dermatitis 21,22 .

General experimental procedures
Melting points were determined on a Kofler hot-stage apparatus and are uncorrected.The NMR spectra of compounds 1 -3 were taken on a Bruker AC 200 ( 1 H: 200 MHz; 13 C: 50.3 MHz) spectrometer.The 1 H (400 MHz) and 13 C (100 MHz) NMR (1D and 2D) spectra of 4 and its acetyl derivative 4a were recorded on a Varian UN 400 spectrometer.HMQC and HMBC experiments were optimized for 1 J CH = 140 Hz and n J CH (n=2 and 3) = 9 Hz, respectively.Mass spectra were obtained with a VG Quattro instrument.FTIR spectra were recorded as a film on a Perkin-Elmer 1500 spectrometer.Column chromatography and on TLC plates were performed using silica gel Merck.

Plant material
Ouratea floribunda (St.Hill) Engl.was collected at an environmentally preserved area near Centro de Pesquisas Morro do Chapéu, Belo Horizonte, Minas Gerais, Brazil, in 1996.Identification of the plant material was performed by Jorge L. Silva by comparison with a herbarium specimen (n o 6944) deposited at the Herbarium José Badini in the Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Minas Gerais State, Brazil.

Extraction and isolation of the constituents
Dried wood material (1334 g) was powdered and sequentially extracted with hexane, EtOAc and MeOH by maceration at room temperature.The hexane extract was evaporated under vacuum to yield 4.3 g of residue.This residue was subjected to chromatography on silica gel column using hexane, dichloromethane and methanol with a gradient of polarity from dichloromethane to methanol.Thirty three fractions of 125 mL each were collected.