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Anais da Academia Brasileira de Ciências (2009) 81(1): 21-28 (Annals of the Brazilian Academy of Sciences) ISSN 0001-3765 www.scielo.br/aabc

New iodine derivatives of flavonol and

, MÁRIO G. DE CARVALHO1, VIRGINIA C. DA SILVA1, TÂNIA M.S. DA SILVA1 2, , CELSO A. CAMARA3 and RAIMUNDO BRAZ-FILHO1 4 1Departamento de Química, ICE, Universidade Federal Rural do Rio de Janeiro, BR-465 km 07 23890-000 Seropédica, RJ, Brasil 2Núcleo Complexo Produtivo de Saúde, IMS-CAT-UFBA, Av. Olívia Flores, 3.000, Candeias 45055-090 Vitória da Conquista, BA, Brasil 3Departamento de Química, Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros s/n Dois Irmãos, 52171-900 Recife, PE, Brasil 4Setor de Química de Produtos Naturais, LCQUI-CCT, Universidade Estadual Norte Fluminense Darcy Ribeiro (UENF) 28013-602 Campos, RJ, Brasil

Manuscript received on November 19, 2007; accepted for publication on November 4, 2008; contributed by RAIMUNDO BRAZ-FILHO*

ABSTRACT The reaction of the flavonol 3,7,30, 40-tetra-O-methylquercetin (1) and of the isoflavone 7,40-di-O-methylgenistein (2) with alkaline iodine in methanol afforded four new iodine derivatives: 8-iodo-5-hydroxy-3,7,30, 40-tetramethoxy- flavone (1a) and 6-iodo-5-hydroxy-3,7,30, 40-tetramethoxyflavone (1b) from 1; 2 afforded a mixture of two com- ) pounds, identified as a racemic mixture of(± -trans-5-hydroxy-2,3,7,40-tetramethoxy-8-iodo-isoflavanone (2a) and ) (± -trans-5-hydroxy-2,3,7,40-tetramethoxy-6,8-diiodo-isoflavanone (2b). The formation of these different products reveals a significant difference involving the chemical interaction between the reactive siteof α, β-unsaturated ke- tones of flavonol and isoflavone under the tested reaction conditions (usingI2/KOH/MeOH). Furthermore, the trans stereoselectivity is noteworthy in the nucleophylic addition of methanol at the isoflavone α, β-unsaturated system. The structures were identified on the basis of spectral data, mainly 1D and 2D NMR and massspectra. Key words: flavonol, isoflavone, iodoflavonoid, iodoisoflavonoid, iodine derivatives.

INTRODUCTION groups to improve those activities have been frequently observed (Dejjermm 1997, Paulo and Mota-Filipe 2006). In previous reports we have described the isolation and The iodination of natural is a procedure that has identification of natural in Solanaceae (Silva been used to obtain some useful intermediates in the syn- 2002, Silva et al. 2004) and Leguminosae (Silva et al. thesis of biflavonoids (Zheng et al. 2004, Ali and Ilyas 2006, 2007), preparation of some derivatives and made 1986). the unambiguous proton and carbon-13 chemical shifts Halogenated derivatives of natural flavonoids are assignments (Carvalho et al. 2006). Citations concern- relatively rare in the literature, particularly with the io- ing the synthesis (Guo-Qiang and Zhong 1997, Zem- dine derivate, whose synthetic utility is largely due to bower and Zhang 1998, Quintin and Lewing, 2004, the increase in reactivity of this particular halogen, as Bekker et al. 1998) and biological importance of flavo- in nucleophilic aromatic substitutions (Yaipakdee and noids and biflavonoids besides incorporation of any Robertson 2001). Iodine derivatives are described in the

*Member Academia Brasileira de Ciências synthesis of biflavonoids, by using expensive reagents Correspondence to: Mário Geraldo de Carvalho difficult to obtain (Rho et al. 2001, Bovicelli et al.2001). E-mail: [email protected]

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22 MÁRIO G. DE CARVALHO et al.

5' 5' 4' 6' 4' OMe 1 6' OMe R 1 8 1 8 9 7 9 MeO 7 O 2 MeO O 2 3' 1' 3' I 1' OMe 2' OMe 2 2' 3 3 6 10 KOH/MeOH 6 10 4 5 4 OMe R 5 OMe 3h/r.t. OH O OOH R=H, R1=I R=I, R1=H 74% as a mixture

1 1 R 8 1 8 MeO 7 9 O 9 2 MeO 7 O 2 OMe 2' 3 I2 2' 1' 3' 3 6 10 6 1' 3' 5 4 R 10 4 4' KOH/MeOH 5 4' OOH 6' OMe 3h/r.t. OH O 6' 5' OMe 5' OMe (+/-) R=H, R1=I (+/-) R=R1=I 85% as a mixture

Fig. 1 – Structure and reactions of flavonoids 1 and 2.

) In this paper we describe the first iodination reac- and (± -trans-5-hydroxy-2,3,7,40-tetramethoxy-6,8-di- tion of two flavonoids (Fig. 1), the flavonol iodo-isoflavanone (2b) in 85% yield. The products struc- 3,7,30, 40-tetramethyl ether (1, 3,7,30, 40-tetra-O-methyl- tures were identified on the basis of spectral data, mainly quercetin, ) isolated from Solanum species (Silva 1D and 2D NMR and mass spectra. The 1D and 2D NMR 2002, Silva et al. 2004) and the isoflavone 7,40-di-O- spectra were also used to the complete 1H and 13C chem- methylgenistein (2, 5-hydroxy-40, 7-dimethoxyisoflavo- ical shift assignments of the four new products. These ne) obtained by selective methylation of bichanin A (5,7- new derivatives of natural flavonoids, 1a, 1b, 2a and dihydroxy-40-methoxyisoflavone) isolated from Andira 2b, can be used as intermediate in synthetic procedure species (Silva et al. 2006, 2007), using iodine as an and to include useful group to study the mechanism of inexpensive and easily available reagent. These flavo- flavonoids biological activity. noids have C-6 or C-8 positions as two sites for electro- phylic substitutions. The functionality of similar nucleus MATERIALS AND METHODS in synthesis of bichalcones and other biflavonoids have GENERAL EXPERIMENTAL PROCEDURE been reported (Ali and Ilyas 1986). This work led us to synthesize four new iodine derivatives: 5-hydroxy- NMR: Bruker DRX-500 (500 MHz for 1H and 125 MHz 8-iodo-3,7,30, 40-tetramethoxyflavone (1a) and the C-6 for 13C) and Bruker AMX-300 (300 MHz for 1H and corresponding regioisomer (1b) from 1 in 74% yield 75 MHz for 13C) were used to obtain the 1D and 2D ) 1 13 and 2 afforded a mixture constituted by (± -trans-5- H and C NMR spectra and Bruker AC-200 was used hydroxy-2,3,7,40-tetramethoxy-8-iodo-isoflavanone (2a) to make the NOEDIFF experiments, in CDCl3 as sol-

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NEW IODINE DERIVATIVES OF FLAVONOIDS 23

OCH3

OCH3 R OCH3

H3CO O OCH3

R1 OCH3 C O OH O C9H9O3 R=I, R1=H 165.055169 R=H, R1=I 165.055169 C19H17IO7 484.001905 484.014040

Fig. 2 – Proposed fragmentation patterns for 1a/1b derivatives based on the HREIMS.

δ ) δ vent and the signals at H 7.24 (CHCl3 and C 77.00 of 2 (Figs. 1 and 3) yielded a yellow gum as a mixture ) (CDCl3 as internal standards. The LRMS were ob- of 2a and 2b (23.8 mg, 85%). tained on a GC-MS Varian Saturn 2000 with ion trap and IE ionization, at 70 eV. The HRESI-MS were recorded RESULTS AND DISCUSSION on a VG 7070E-HF spectrometer using methanol:H2O, The 3,7,30, 40-tetramethylquercetin (1) af- Ar as CAD, DE 20 eV for MS and 45 eV for MS-MS forded a mixture of 8-iodo-3,7,30, 40-tetramethylquer- using methanol:H2O/formic acid in positive mode. The cetin (1a) and 6-iodo-3,7,30, 40-tetramethylquercetin reactions were monitored by aluminum-backed silica gel (1b) in a 3(1a):1(1b) ration, as determined by inspec- λ TLC plates and visualized under UV ( max 254 nm) or tion of 1H NMR data. The reaction afforded a mixture using 1% AlCl3 in ethanol as revealing spray solution. of regioisomers 1a and 1b involving the C-8 and C-6 positions, respectively, reflecting the higher reactivity SYNTHESISOFTHE COMPOUNDS of the C-8 position under the experimental conditions Derivative 2 (Fig. 1) was prepared by treating a methanol (Fig. 1). The HREIMS of 1a and 1b mixture gave + / solution of biochanin A (40-O-methylgenistein, 50.0 mg) [M] at m z 484.01404 corresponding to the molecu- / with ethereal diazomethane solution followed by sol- lar formula C19H17IO7 (Calcd. for C19H17IO7 m z / vent evaporation (Silva et al. 2006, 2007). The natural 484.00190). From the molecular ion m z 484.014040 / flavonoid 1 (Fig. 1) (Silva 2002) (20.0 mg, 0.056 mMol) was obtained a fragment of m z 165.055169 as base and derivative 2 (28.0 mg, 0.093 mMol), were dissolved peak corresponding to the fragment involving the B- in 10 mL of methanol, mixed with potassium hydroxide ring (Fig. 2). (25.0 mg in each reaction), under continuous stirring, The 1H NMR spectrum of 1a/1b mixture showed and to this mixture iodine was added in small portions signals of 1a (component present in major proportion) to slight excess. The solutions were kept at room tem- in the aromatic region corresponding to four hydrogen δ perature under stirring for 3h. Aliquots of the reaction atoms and the hydrogen-bonded absorption at H 12.92 δ mixture were periodically analyzed by TLC until com- (s) of hydroxyl group at C-5. The singlet at H 6.42 plete reaction. The organic solvents were removed under was attributed to H-6 at A-ring. The chemical shifts, reduced pressure, and the remaining aqueous solution the multiplicity and the coupling constants values (J) of δ δ was extracted with dichloromethane (3 × 15 mL). The the signals in H 8.06 (dd, J=1.8 and 8.7 Hz, H-60), H combined organic solutions were dried with anhydrous 7.98 (d, J=1.8 Hz, H-20), and 7.03 (d, J=8.7 Hz, H-50) sodium sulfate and the solvent was evaporated under re- allowed to recognize the ABC system of B-ring with sub- duced pressure. Reaction of 1 (Fig. 1) yielded a brown- stitutions in the positions 30 and 40 (Table I). The 2D 1H- yellowish gum (1a + 1b, 20.0 mg, 74%) and the reaction 1H-COSY confirmed the interactions spin-spin for these

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24 MÁRIO G. DE CARVALHO et al.

H3COH H3COH I

H CO O O O OCH3 O H3CO O OCH3 3 I2 I OCH3 p.t. OCH3 KOH/CH3OH Ph p.t. Ph R 3h, r.t. I Ph OH O O O O OH O OCH3 (+) R=H (+) R=I OCH3

Fig. 3 – Proposed pathway to the observed trans stereochemistry in isoflavone iodination.

I 8 H3CO 9 O 2 OCH3 I 7 H 3 6 4 H3CO O R 10 2' 5 1' H2C=O 3' OCH3 OHO 4' 6' O R1 Rt = 19 min. 5' OCH3 OH O OCH3 C19H19IO7 R,R1 = H, OMe .+ M 486 (100%) 456 (40%) I H3CO O H OCH3 H OCH3 C H C OH O 3 H CO 3 O O OCH3 OCH3 194 (90%) 179 (40%)

I 8 OCH3 3 2' H3CO 9 O 2 1' 7 H H3CO 3' H 2' 3 6 4 1' 3' 4' O 10 6' 5 O OCH3 OCH3 4' 5' O 6' 5' OCH3 151 (25%)

Fig. 4 – Proposed fragmentation patterns for 2a (only peaks classified as principals).

δ δ δ hydrogen atoms. These data and the singlet between C-5 ( C 163.34), C-7 ( C 163.99), C-8 ( C 60.98), and δ δ 3.91-4.0 ppm corresponding to four methoxyl groups C-10 ( C 106.15) with H-6 ( H 6.42). The heteronu- δ δ allowed us to characterize the product as a 5-hidroxy- clear interactions of OH-5 ( H 12.92) with both C-5 ( C 13 2 ) δ 3 ) tetramethoxy-flavonol. The C NMR-PENDANT spec- 163.34, JC H and CH-6 ( C 95.45, JC H were used to trum revealed signals for nineteen carbons, being four characterize definitively the 1a structure. Other correla- methynes, four methoxyl groups, and eleven quarternary tions revealed by HMBC are shown in Table I. The iodine 1 13 1 carbon atoms. The 2D H- C-COSY- JC H (HMQC) derivative 1b was also characterized by same procedure, spectrum was used to recognize the direct correlations which revealed the data described in Table I. 1 ) ( JC H of the hydrogen and carbon atoms corresponding However, the same reaction using the isoflavone (2) to methynes and methoxyl groups (Table I). The correct yielded different products when compared with those ob- position of methyne carbons was confirmed by heteronu- tained of flavonol 1, by the presence of two additional 2 3 ) clear interactions at long range ( JC H and JC H of the methoxyl groups located at CH-2 and C-3 of the enone

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NEW IODINE DERIVATIVES OF FLAVONOIDS 25

I OCH H3CO O 3 I H H3CO O OCH3 I OCH3 OCH OHO 3 I OH O OCH3 OCH3 581 (10%) Rt = 25.3 min, C19H18I2O7 M.+ 612 (100%) I I OCH 2 H3CO O H3CO O R OCH O 2 I I R OH O OH O 1 O OCH3 OCH3 552 (10%) R, R1 = H, OCH3 582 (10%)

I RDA H OCH3 H3CO O

O H CO IC + 3 O OH OCH3 418 (9%) 194 (90%)

H CO O 3

OCH3 151 (40%)

Fig. 5 – Proposed fragmentation patterns for 2b (only peaks classified as principals). system (Figs. 1 and 3). The CG-LREIMS of the mix- additional peaks in the mass spectra m/z (%): 2a: 486 ture containing the compounds 2a and 2b showed two (100, C19H19O7I); 456 (40, M-30), 194 (90), 179 (40), / ) peaks [TR 19.0 min (58.14%) and TR 25.3 (41.86%)] 151 (25) Figure 4; 2b: m z 612 (100, C19H18O7I2 , 582 and the corresponding molecular ions peaks identified at (10), 552 (10), 418 (9), 194 (90), 151 (40); been 194 / ) m z 486 (100%, 2a) and 612 (100%, 2b), as summa- = H3COCH=COCH3(C6H4-OCH3 by RDA, Figure 5. rized in Figures 4 and 5. Besides these data, the analy- The data of 1H and 13C NMR ({1H} and DEPT) 1D- and sis of 1H and 13C NMR let us to identify the chemical 2D (1Hx1H-COSY, HMQC and HMBC) spectra analy- shifts represented by two close values (Table I), suggest- sis are described in Table I. The NOEDIFF experiments ing similar structures compatible with the presence of were also used to confirm these structures, contribut- these two enantiomeric pairs (2a and 2b). The detailed ing to complete 1H and 13C NMR assignments (Table mass spectral data analysis and the NOEDIFF experi- I) and to identify the relative configuration of C-2 and δ ments justified both similar structures with two addi- C-3. The irradiations at H 3.63 (H3CO-2, 2a and 2b) δ tional methoxyl groups with one and two incorporated yield NOE at H-2 [5.22 (2a) and H 5.18 (2b)], irradi- δ iodine in 2a and 2b, respectively. These structures and ation at 3.19 (H3CO-3, 2b) yield NOE at H-2 ( H 5.18, δ , δ locations of those groups were recognized by analysis of 2b), HO-5 ( H 12.17) and at H-20 60( H 7.42); irradi-

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26 MÁRIO G. DE CARVALHO et al. ) , ) ) , δ ± δ ± , δ δ δ , δ δ ∗

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NEW IODINE DERIVATIVES OF FLAVONOIDS 27

δ δ ation at H 3.86 (H3CO-400 of 2a) yielded NOE at H (CENAUREMN) for the 500 MHz NMR spectra and , δ 6.98 (H-30 50) and at H 3.95 (H3CO-7 of 2a) yielded Instituto de Química – Universidade Federal do Rio de δ NOE at 6.23 (H-6); irradiation at H 3.21 (H3CO-3, 2a) Janeiro (IQ-UFRJ) for the 300 MHz NMR spectra. δ δ , yield NOE at H 5.22 (H-2), H 7.39 (H-20 60) and HO- δ RESUMO 5 ( H 12.81); no signal was observed in the spectrum δ , obtained from irradiation at H 3.97 (H3CO-7 of 2b); A reação do flavonol 3,7,30 40-tetra-O-metilquercetina (1) e δ , on the other hand a doublet at H 6.96 (H-30 50) was da isoflavona 7,40-di-O-metilgenisteina (2) com iodo/KOH em δ observed in the spectrum obtained by irradiation at H metanol forneceu como produto quatro derivados iodados: 8- , 3.87 (H3CO-40 of 2b). Thus the NOE observed at H-2 iodo-5-hidroxi-3,7,30 40-tetrametoxiflavona (1a) e 6-iodo-5- , and H-3 with irradiation at the H3CO-2 was used to jus- hidroxi-3,7,30 40-tetrametoxiflavona (1b) a partir da iodação tify the trans correlation relationship between methoxyl de 1; a partir de 2 foi obtida uma mistura racêmica composta ( ) groups at 2 and 3 positions, according to the proposed de ± -trans-5-hidroxi-2,3,7,40-tetrametoxi-8-iodo-isoflavano- ( ) mechanism summarized in Figure 3. The detailed anal- na (2a) e ± -trans-5-hidroxi-2,3,7,40-tetrametoxi-6,8-diiodo- ysis of 1H and 13C NMR (1D and 2D) spectra let us isoflavanona (2b). A formação destes diferentes produtos re- to make the complete data assignments of these new vela a significante diferença envolvendo a interação química derivatives (Table I). entre o sitio reativo de cetonas α, β-insaturadas de flavonol In this work we rationalize trans stereoselectivity e de isoflavonas nas condições experimentais testadas (usando α, β observed at the -enone system of 2 as a well estab- I2/KOH/MeOH). Além disso, ressalta-se a estereosseletividade lished addition of methanol to an intermediate halonium trans na adição de metanol ao sistema α, β-insaturado da iso- species (Bateman et al. 1983), followed by alkoxide flavona. As estruturas foram identificadas com análise nosda- substitution of the resulting β-alkoxy-(or β-hydroxy-)- dos espectrométricos de RMN 1D e 2D e massas. α-halo derivative intermediate (Bird et al. 1983). Pre- Palavras-chave: flavonol, isoflavona, iodoflavonóide, iodo- sumably, solvolytic conditions in methanol drives the isoflavonóide, derivados de iodo. incipient benzylic carbenium formed to be anchimeric REFERENCES assisted by a methoxy group (Smith et al. 1965), thus explaining the trans selectivity observed in the products. ALI SM AND ILYAS M. 1986. Biomimetic approach to bi- The reactions performed in the present work are of ex- flavonoids: oxidative coupling of20-hidroxychalcones ploratory nature, carried out to ascertain experimental with I2 in alkaline methanol. J Org Chem 51: 5415–5417. conditions and to ensure the observed products. BATEMAN G,BROWN BR,CAMPBELL JB,COTTON CA, As far as we know, this is the first work that de- JOHNSON P,MULQUEEN P,O’NEILL D,SHAW MR, SMITH RA AND TROKE JA. 1983. Synthesis and reac- scribes the incorporation of nuclear iodine atoms in fla- tions of 4-aryloxy-flavanes. J Chem Soc Perkin Trans 12: vonoids 1 and 2 along with the stereoselective trans in- 2903–2912. corporation of two methoxyl groups at the 2 and 3 posi- BEKKER R,BRANDT EV AND FERREIRA D. 1998. Struc- tions of compound 2. ture and Stereochemistry of the first isoflavanone-benzo- furanone biflavonoids. Tetrahedron Lett 39: 6407–6410. ACKNOWLEDGMENTS BIRD TGC,BROWN BR,STUART IA AND TYRRELL AWR. The authors are grateful to Conselho Nacional de Desen- 1983. Reactions of flav-2-enes and flav-2-en-4-ones (fla- vones). J Chem Soc Perkin Trans 1: 1831–1846. volvimento Científico e Tecnológico (CNPq), Coorde- BOVICELLI P,MINCIONE E,ANTONIOLETTI R,BERNINI nação de Aperfeiçoamento de Pessoal de Nível Superior R AND COLOMBARI M. 2001. Selective halogenation of (CAPES), Fundação Carlos Chagas Filho de Amparo à aromatics by dimethyldioxirane and halogen ions. Synth Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Pro- Commun 31: 2955–2963. grama de Apoio Científico e Tecnológico/Financiadora CARVALHO MG DE, GOMES MS DA R,PEREIRA AHF, de Estudos e Projetos (PADCT/FINEP) for fellowships DANIEL JF DE S AND SCHRIPSEMA J. 2006. Carbon- and financial support. We also thank Centro Nordestino 13 and proton NMR assignments of a new agathisflavone de Aplicação e Uso da Ressonância Magnética Nuclear derivative. Magn Reson Chem 44: 35–37.

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