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

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1 J. Braz. Chem. Soc., Vol., No. 2, 4-47, Printed in Brazil c 2000 Soc. Bras. Química 00-0 $ Article Chemical Constituents from uratea floribunda: Complete and C NMR Assignments of Atranorin and its New Acetyl Derivative Mário G. de Carvalho a*, Geizi J. A. de Carvalho a and Raimundo Braz-Filho b a Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, 2-70, Seropédica - RJ, Brazil b Setor de Química de Produtos Naturais-LCQUI- CCT, Universidade Estadual do Norte Fluminense, 20-20, Campos - RJ, Brazil. fracionamento cromatográfico do extrato hexânico da madeira de uratea floribunda (chnaceae) forneceu, além dos triterpenos friedelina (), friedelanol (2) e lupeol (), o depsídeo atranorina (4). As estruturas das substâncias isoladas foram determinadas através da análise dos dados espectroscópicos de RMN e C, 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 uni- e bidimensionais de RMN ( - CSY e NESY, - C MBC e MQC) e está sendo descrita pela primeira vez na literatura. Chromatographic fractionation of the hexane extract from the wood of uratea floribunda (chnaceae) afforded friedelin (), friedelanol (2), lupeol () and the depside atranorin (4). The structure elucidation of the isolated compounds was performed by spectrometric analysis involving comparison with literature data. The unambiguous assignments of and C NMR data of atranorin 4 and its acetyl derivative 4a are reported for the first time and involved - homonuclear (CSY and NESY) and - C heteronuclear (MQC and MBC) NMR experiments. Keywords: uratea floribunda, chnaceae, atranorin, monoacetylatranorin Introduction The genus uratea (chnaceae) comprises ca 00 tropical species occurring mainly in South America. Some species of this genus have been shown to possess antiviral (uratea lucens, from Panama) 2, antimicrobial (. parviflora, from Brazil) as well as pain relief (. reticulata, from Guinea) activities 4. The species. 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. The chemical study of this genus has led to the isolation of a biscatequin (uratea sp.), a cyanidin and a flavonoid (. affinis) 7, biisoflavanones, an isoflavone and triterpenes (. hexasperma), biflavones (. spectabilis) and recently we reported the isolation of biflavones, flavonoid glycosides, chloroisoflavonoids, norisoprenoids, lignans, a diterpene 0, triterpenes and steroids from. semiserrata. As part of our chemical and pharmacological studies of Brazilian plants we reported the investigation of some uratea species -. 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 2. In this first study of. floribunda we report the presence of known triterpenes ( - ) 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 and C NMR data of atranorin 4 and of its monoacetyl derivative 4a are reported for the first time and involved - homonuclear (CSY and NESY) and - C heteronuclear (MQC and MBC) NMR experiments. Results and Discussion The hexane extract of the dry wood of uratea floribunda (St. ill) Eng. was submitted to chromatography on a

2 44 Carvalho et.al J. Braz. Chem. Soc R R 2 R, R 2 = 2 R =, R 2 = 4 R ' 7 ' 2 7' ' ' ' 4 R= 4a R=Ac ( observed NE) silica gel column to afford three triterpenes - (.4 x 0-2 % of dry wood weight) and the depside 4 (.0 x 0-4 % of dry wood weight). The known triterpenes friedelin (), friedelanol (2) and lupeol () were identified by their and C NMR spectral data analysis and comparison with literature values -. The molecular formula of 4 was deduced as C by a combination of LRMS (m/z 7 [M] +, %, C ), and C (BBD and DEPT) NMR spectra analysis. Its NMR spectrum showed the presence of 4b singlet signals at δ 2.07, 2., 2.7 and.7 corresponding to three methyl groups bonded to sp 2 carbons and one methoxyl group, respectively. This spectrum also revealed six deshielded singlet signals corresponding to two aromatic hydrogens (δ. and.0), a formyl hydrogen (δ 0.0) and three quelated hydroxyl groups (δ.4, 2.4 and 2.4). The comparative analysis of BBD, DEPT and MQC NMR spectra (Table ) showed twelve quaternary sp 2 carbon signals, three C (δ C 2.,. and.) and four C (δ C.4, 24.0, 2. and 2.) consistent with the data obtained by NMR analysis. The chemical shifts of quaternary sp 2 carbons [δ C 02., 0., 0.,.7,., 2.0, 2.4, 2., 7.,.,.7 (C=), and 72.2 (C=)] were used to propose two aromatic rings sustaining five substituents (vide infra) and one hydrogen atom [δ.0 (s) and. (s)] each. The MBC experiments showed 2, J C long-range correlations (Table ) between the -' (δ 2.) and carbons C-' (δ C., 2 JC), C-' (δ C., J C ) and C- (δ C 0., J C ); between - (δ 2.07) and C- (δ C.7, 2J C ), C-' (δ C 2., J C ) and C-' (δ C 2.0, J C ); and between -' (δ.0) and C-' (δ C 2.0, 2 J C ), (C- (δ C.7, J C ) and C- (δ C 0., J C ). Therefore, the two methyl groups C - and C -' are at the same ring. The other cross peaks (Table ) are also in agreement with the location of one methoxycarbonyl and one hydroxyl group in this same aromatic ring. These deductions and Table. (400 Mz) and C (00 Mz) NMR spectral data of atranorin (4) and its acetyl derivative 4a, in CDCl and TMS as internal standard. 4 4a C/ δ C (C X ) a,b δ (n, m) a MBC c δ C (C X ) a,b δ (n, m) a MBC c 02.(C) - -2; -, 02.7(C) (C) - -2.(C) - 0.(C) - -2,4; - 0.(C) - -2,4; (C) - -; -4 7.(C) (C) 0. (, s) -4; - 2.(C) 0.(, s) - 2.4(C) (C) (C) - -.4(C) - -.(C) 0.0 (, s) -.(C) 0.40 (, s) - 2.(C ) 02.7 (, s) - 2.(C ) 02. (, s) 2.0(C) - - ; - 4.7(C) - - ; - 2.7(C) - - ; -,.(C) (C) - - ; - 4.4(C) (C) - - ; -, 24.(C) - -.(C) - -.(C) - -.(C) 0.0 (, s) - 2.(C) 0. (, s) (C) - C-7.(C) - C-7.4(C ) (, s) - 0.(C ) 02.0 (, s) (C ) 02. (, s) - 20.(C ) 02. (, s) - C- 2.(C ) 0.7 (, s) - 2.(C ) 0. (, s) - C= (C) - C-C= C (C ) 02.0 (, s) (, s) (, s) (, s) (, s) (, s) a Number of hydrogens bonded to carbons were deduced by comparative analysis of BBD and DEPT C NMR experiments; b Assignments aided by NESY and MQC experiments; c MBC J C value = z.

3 Vol. No. 2, 2000 Chemical Constituents from uratea floribunda 4 the peaks at m/z (00 %), 4 (0 %) and 7 ( %) observed in the mass spectrum (Scheme ) are in agreement with the presence of a methyl 2-hydroxy-,-dimethylbenzoate moiety. The nuclear verhauser enhancements observed in - -NESY spectrum was used to confirm the location of -' (δ 2.) ortho to -' (δ.0). The remaining 2, J C cross peaks observed in the MBC spectrum were used to establish the correlation between the signals of two hydroxyl, -2 (δ 2.4) with C-2 (δ C., 2J C ) and C- (δ C 0., J C ), -4 (δ 2.4) with C- (δ C 0., J C ), C-4 (δ C 7., 2 J C ) and C- (δ C 2., J C ), and to assign the chemical shifts for C-2, C-, C-4 and C- (Table ). The observed long-range correlation involving the remaining methyl group C - (δ C 2.) and the aromatic hydrogen - (δ.) led to the assignments of C- and C- (Table ). The NE observed between - (δ.) and - (δ 2.7) in - NESY experiment was also used to confirm the location of - ortho to -. These data and the peaks at m/z (00 %), (20%), 7 ( %) and (40 %) observed in the mass spectrum (Scheme ) are in accordance with the presence of a 2,4-dihydroxy--aldehyde--methyl-benzoate moiety. Thus, these data allowed to deduce the structure of 4, a natural product classified as depside and known as atranorin, isolated from lichens. The structure was previously determined only by NMR data and confirmed through synthesis -. The acetylation of 4 at 0 o C yielded the hydrolysis product, methyl 2-hydroxy-4-hydroxy-,-dimethylbenzoate (4b). The same acetylation carried out at room temperature gave the new monoacetate 4a, whose structure was confirmed by complete and C NMR spectra assignment (Table ). As was expected, the chemical shift of -' was shifted from δ.0 in 4 to δ. in 4a, with its location at ortho or at para positions and, in accordance with the presence of -' para in relation to the '-hydroxyl group in 4. This is the first report for C NMR data assignments for the depside 4 [-formyl-2,4-dihydroxy--methylbenzoic acid -hydroxy-4-(methoxycarbonyl)-2,-dimethylphenyl ester] and the derivative 4a [-formyl-2,4-dihydroxy-- methylbenzoic acid -acetoxy-4-(methoxycarbonyl)-2,- dimethylphenyl ester]. 4 ' 7 ' 2 + ' 7' ' ' m/z 7 ([M+] +, %) ' ' ' 7' ' and/or ' m/z (00 %) C 2 m/z 7 ( %) C m/z (20 %) C 2 ' ' ' 7' ' ' m/z 4 (0 %) C C m/z (40 %) Scheme. Proposed fragmentation mechanisms of 4 (only peaks classified as principal).

4 4 Carvalho et.al J. Braz. Chem. Soc 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 C 2, C and groups, frequently found in lichens, it seems that the wood of. floribunda used in this study was contaminated. Among the aromatic lichen compounds, atranorin is the most frequent allergenic agent responsible for airborne contact dermatitis 2,22. Experimental General experimental procedures lting points were determined on a Kofler hot-stage apparatus and are uncorrected. The NMR spectra of compounds - were taken on a Bruker AC 200 ( : 200 Mz; C: 0. Mz) spectrometer. The (400 Mz) and C (00 Mz) NMR (D and 2D) spectra of 4 and its acetyl derivative 4a were recorded on a Varian UN 400 spectrometer. MQC and MBC experiments were optimized for J C = 40 z and n J C (n=2 and ) = z, respectively. Mass spectra were obtained with a VG Quattro instrument. FTIR spectra were recorded as a film on a Perkin-Elmer 00 spectrometer. Column chromatography and on TLC plates were performed using silica gel rck. Plant material uratea floribunda (St. ill) Engl. was collected at an environmentally preserved area near Centro de Pesquisas Morro do Chapéu, Belo orizonte, Minas Gerais, Brazil, in. Identification of the plant material was performed by Jorge L. Silva by comparison with a herbarium specimen (n o 44) deposited at the erbarium José Badini in the Instituto de Ciências Exatas e Biológicas, Universidade Federal de uro Preto, Minas Gerais State, Brazil. Extraction and isolation of the constituents Dried wood material (4 g) was powdered and sequentially extracted with hexane, EtAc and by maceration at room temperature. The hexane extract was evaporated under vacuum to yield 4. 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 2 ml each were collected. Fractions - yielded a mixture of and 2 (20 mg, m. p o C). Fractions -0 yielded (0 mg, m. p. 0- o C). Fractions -4 gave 4 (2 mg, m. p. -2 o C). Atranorin (4) Colorless crystals from hexane, m. p. -2 o C. IR (KBr) ν max (cm - ): 40, 770, 70, 0, 0, 20, 4. (400 Mz, CDCI ) and C (00 Mz, CDCl ) NMR: Table. CIMS: Scheme. Acetylation of atranorin (4) Compound 4 ( mg) was dissolved in pyridine (.0 ml) and acetic anhydride (.0 ml), and the solution was allowed to stand for 24 h at room temperature. Usual work-up yielded 4a (gum, 7 mg), amorphous powder from hexane, m. p. 0-0 o C, IR (film) ν max (cm - ): 772, 7, 4, 72, 20, 0. NMR (400 Mz, CDCl ) and C NMR (00 Mz, CDCI ): Table. CIMS m/z (rel. int.): 47 ([M + ] +, 2), 2 (20), 22 (2), 207 (00), 7 (7), 7 (2). The acetylation of 4 at 0 o C yielded methyl 2-acetoxy- 4-hydroxy-,-dimethylbenzoate (4b): gum, NMR (400 Mz, CDCl ) δ :.2 (s, -),. (s, -7), 2.0 (s, ),. (s, ). Acknowledgements The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for research fellowships and for grants to G. J. A. de C.. We thank Mr. J. L. Silva and Dr. A. A. Werle (Universidade Federal de uro Preto, Minas Gerais, Brazil) for the collection and identification of plant material. We thank Dr D. G. I. Kingston, Virginia Polytechnic Institute and State University-USA, for mass, and C NMR spectrometers facilities and Dr. Victor Runjanek for reading the manuscript. References. eywood, V.. In Flowering Plants of the World; xford University Press; London, 7, p Roming, T. L.; Weber, N. D.; Murray, B. K.; North, J. A.; Wood, J. C.; ughs, A. G.; Cates, R. G. Phytother. Res. 2,,.. Paulo, M. Q.; Lima, E..; Maia, R. F.; Filho, X. L. Chin Chiao I Sheng Tsa Chin,,. 4. Vasileva, B. Plantes dicinales De Guine; Conakry, Rep. De Guine, Moscow, Univ. Moscow-USSK,.. Felicio, J. D.; Gonçalvez, E.; Braggio, M. M.; Costantino, L.; Albasini, A.; Lins, A. P. Planta d.,, 27.

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