New iodine derivatives of flavonol and isoflavone

CHEMICAL SCIENCES

New iodine derivatives of flavonol and isoflavone

Mário G. de Carvalho^I; Virginia C. da Silva^I; Tânia M.S. da Silva^{I, II}; Celso A. Camara^III; Raimundo Braz-Filho^{I, IV}

^IDepartamento de Química, ICE, Universidade Federal Rural do Rio de Janeiro, BR-465 km 07 23890-000 Seropédica, RJ, Brasil

^IINúcleo Complexo Produtivo de Saúde, IMS-CAT-UFBA, Av. Olívia Flores, 3.000, Candeias 45055-090 Vitória da Conquista, BA, Brasil

^IIIDepartamento de Química, Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros s/n Dois Irmãos, 52171-900 Recife, PE, Brasil

^IVSetor de Química de Produtos Naturais, LCQUI-CCT, Universidade Estadual Norte Fluminense Darcy Ribeiro (UENF) 28013-602 Campos, RJ, Brasil

^{Correspondence to}

ABSTRACT

The reaction of the flavonol 3,7,3', 4'-tetra-O-methylquercetin (1) and of the isoflavone 7,4'-di-O-methylgenistein (2) with alkaline iodine in methanol afforded four new iodine derivatives: 8-iodo-5-hydroxy-3,7,3', 4'-tetramethoxy- flavone (1a) and 6-iodo-5-hydroxy-3,7,3', 4'-tetramethoxyflavone (1b) from 1; 2 afforded a mixture of two compounds, identified as a racemic mixture of (±)-trans-5-hydroxy-2,3,7,4'-tetramethoxy-8-iodo-isoflavanone (2a) and (±)-trans-5-hydroxy-2,3,7,4'-tetramethoxy-6,8-diiodo-isoflavanone (2b). The formation of these different products reveals a significant difference involving the chemical interaction between the reactive site of α, β-unsaturated ketones of flavonol and isoflavone under the tested reaction conditions (using I₂/KOH/MeOH). Furthermore, the trans stereo selectivity 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 mass spectra.

Key words: flavonol, isoflavone, iodoflavonoid, iodoisoflavonoid, iodine derivatives.

RESUMO

A reação do flavonol 3,7,3',4'-tetra-O-metilquercetina (1) e da isoflavona 7,4'-di-O-metilgenisteina (2) com iodo/KOH em metanol forneceu como produto quatro derivados iodados: 8-iodo-5-hidroxi-3,7,3',4'-tetrametoxiflavona (1a) e 6-iodo-5-hidroxi-3,7,3',4'-tetrametoxiflavona (1b) a partir da iodação de 1; a partir de 2 foi obtida uma mistura racêmica composta de (±)-trans-5-hidroxi-2,3,7,4'-tetrametoxi-8-iodo-isoflavanona (2a) e (±)-trans-5-hidroxi-2,3,7,4'-tetrametoxi-6,8-diiodo-isoflavanona (2b). A formação destes diferentes produtos revela a significante diferença envolvendo a interação química entre o sitio reativo de cetonas α, β-insaturadas de flavonol e de isoflavonas nas condições experimentais testadas (usando I₂/KOH/MeOH). Além disso, ressalta-se a estereosseletividade trans na adição de metanol ao sistema α, β-insaturado da isoflavona. As estruturas foram identificadas com análise nos dados espectrométricos de RMN 1D e 2D e massas.

Palavras-chave: flavonol, isoflavona, iodoflavonóide, iodoisoflavonóide, derivados de iodo.

INTRODUCTION

In previous reports we have described the isolation and identification of natural flavonoids in Solanaceae (Silva 2002, Silva et al. 2004) and Leguminosae (Silva et al. 2006, 2007), preparation of some derivatives and made the unambiguous proton and carbon-13 chemical shifts assignments (Carvalho et al. 2006). Citations concerning the synthesis (Guo-Qiang and Zhong 1997, Zembower and Zhang 1998, Quintin and Lewing, 2004,Bekker et al. 1998) and biological importance of flavonoids and biflavonoids besides incorporation of any groups to improve those activities have been frequently observed (Dejjermm 1997, Paulo and Mota-Filipe 2006). The iodination of natural flavones is a procedure that has been used to obtain some useful intermediates in the synthesis of biflavonoids (Zheng et al. 2004, Ali and Ilyas 1986).

Halogenated derivatives of natural flavonoids are relatively rare in the literature, particularly with the iodine derivate, whose synthetic utility is largely due to the increase in reactivity of this particular halogen, as in nucleophilic aromatic substitutions (Yaipakdee and Robertson 2001). Iodine derivatives are described in the synthesis of biflavonoids, by using expensive reagents difficult to obtain (Rho et al. 2001, Bovicelli et al. 2001).

In this paper we describe the first iodination reaction of two flavonoids (Fig. 1), the flavonol quercetin 3,7,3',4'-tetramethyl ether (1, 3,7,3',4'-tetra-O-methylquercetin, retusin) isolated from Solanum species (Silva 2002, Silva et al. 2004) and the isoflavone 7,4'-di-O-methylgenistein (2, 5-hydroxy-4', 7-dimethoxyisoflavone) obtained by selective methylation of bichanin A (5,7-dihydroxy-4'-methoxyisoflavone) isolated from Andira species (Silva et al. 2006, 2007), using iodine as an inexpensive and easily available reagent. These flavonoids have C-6 or C-8 positions as two sites for electrophylic substitutions. The functionality of similar nucleus in synthesis of bichalcones and other biflavonoids have been reported (Ali and Ilyas 1986). This work led us to synthesize four new iodine derivatives: 5-hydroxy-8-iodo-3,7,3',4'-tetramethoxyflavone (1a) and the C-6corresponding regioisomer (1b) from 1 in 74% yield and2 afforded a mixture constituted by (±)-trans-5-hydroxy-2,3,7,4'-tetramethoxy-8-iodo-isoflavanone (2a) and (±)-trans-5-hydroxy-2,3,7,4'-tetramethoxy-6,8-diiodo-isoflavanone (2b) in 85% yield. The products structures were identified on the basis of spectral data, mainly 1D and 2D NMR and mass spectra. The 1D and 2D NMR spectra were also used to the complete ¹H and ¹³C chemical shift assignments of the four new products. These new derivatives of natural flavonoids, 1a, 1b, 2a and 2b, can be used as intermediate in synthetic procedure and to include useful group to study the mechanism of flavonoids biological activity.

MATERIALS AND METHODS

GENERAL EXPERIMENTAL PROCEDURE

NMR: Bruker DRX-500 (500 MHz for ¹H and 125 MHz for ¹³C) and Bruker AMX-300 (300 MHz for ¹H and 75 MHz for ¹³C) were used to obtain the 1D and 2D ¹H and ¹³C NMR spectra and Bruker AC-200 was used to make the NOEDIFF experiments, in CDCl₃ as solvent and the signals at δ_H 7.24 (CHCl₃) and δ_C 77.00 (CDCl₃) as internal standards. The LRMS were obtained on a GC-MS Varian Saturn 2000 with ion trap and IE ionization, at 70 eV. The HRESI-MS were recorded on a VG 7070E-HF spectrometer using methanol:H₂O, Ar as CAD, DE 20 eV for MS and 45 eV for MS-MS using methanol:H₂O/formic acid in positive mode. The reactions were monitored by aluminum-backed silica gel TLC plates and visualized under UV (λ_max 254 nm) or using 1% AlCl₃ in ethanol as revealing spray solution.

SYNTHESIS OF THE COMPOUNDS

Derivative 2 (Fig. 1) was prepared by treating a methanol solution of biochanin A (4'-O-methylgenistein, 50.0 mg) with ethereal diazomethane solution followed by solvent evaporation (Silva et al. 2006, 2007). The natural flavonoid 1 (Fig. 1) (Silva 2002) (20.0 mg, 0.056 mMol) and derivative 2 (28.0 mg, 0.093 mMol), were dissolved in 10 mL of methanol, mixed with potassium hydroxide (25.0 mg in each reaction), under continuous stirring, and to this mixture iodine was added in small portions to slight excess. The solutions were kept at room temperature under stirring for 3h. Aliquots of the reaction mixture were periodically analyzed by TLC until complete reaction. The organic solvents were removed under reduced pressure, and the remaining aqueous solution was extracted with dichloromethane (3 × 15 mL). The combined organic solutions were dried with anhydrous sodium sulfate and the solvent was evaporated under reduced pressure. Reaction of 1 (Fig. 1) yielded a brown-yellowish gum (1a + 1b, 20.0 mg, 74%) and the reaction of 2 (Figs. 1 and ³) yielded a yellow gum as a mixture of 2a and 2b (23.8 mg, 85%).

RESULTS AND DISCUSSION

The flavonoid 3,7,3',4'-tetramethylquercetin (1) afforded a mixture of 8-iodo-3,7,3',4'-tetramethylquercetin (1a) and 6-iodo-3,7,3',4'-tetramethylquercetin(1b) in a 3(1a):1(1b) ration, as determined by inspection of ¹H NMR data. The reaction afforded a mixture of regioisomers 1a and 1b involving the C-8 and C-6 positions, respectively, reflecting the higher reactivity of the C-8 position under the experimental conditions (Fig. 1). The HREIMS of 1a and 1b mixture gave[M]⁺ at m/z 484.01404 corresponding to the molecular formula C₁₉H₁₇IO₇ (Calcd. for C₁₉H₁₇IO₇m/z 484.00190). From the molecular ion m/z 484.014040 was obtained a fragment of m/z 165.055169 as base peak corresponding to the fragment involving the B-ring (Fig. 2).

The ¹H NMR spectrum of 1a/1b mixture showed signals of 1a (component present in major proportion) in the aromatic region corresponding to four hydrogen atoms and the hydrogen-bonded absorption at δ_H 12.92 (s) of hydroxyl group at C-5. The singlet at δ_H 6.42 was attributed to H-6 at A-ring. The chemical shifts, the multiplicity and the coupling constants values (J) of the signals in δ_H 8.06 (dd, J=1.8 and 8.7 Hz, H-6'), δ_H 7.98 (d, J=1.8 Hz, H-2'), and 7.03 (d, J=8.7 Hz, H-5') allowed to recognize the ABC system of B-ring with substitutions in the positions 3' and 4' (Table I). The 2D ¹H-¹H-COSY confirmed the interactions spin-spin for these hydrogen atoms. These data and the singlet between 3.91-4.0 ppm corresponding to four methoxyl groups allowed us to characterize the product as a 5-hidroxy-tetramethoxy-flavonol. The ¹³C NMR-PENDANT spectrum revealed signals for nineteen carbons, being four methynes, four methoxyl groups, and eleven quarternary carbon atoms. The 2D ¹H-¹³C-COSY-¹J_CH (HMQC) spectrum was used to recognize the direct correlations (¹J_CH) of the hydrogen and carbon atoms corresponding to methynes and methoxyl groups (Table I). The correct position of methyne carbons was confirmed by heteronuclear interactions at long range (²J_CH and ³J_CH) of the C-5 (δ_C 163.34), C-7 (δ_C 163.99), C-8 (δ_C 60.98), and C-10 (δ_C 106.15) with H-6 (δ_H 6.42). The heteronuclear interactions of OH-5 (δ_H 12.92) with both C-5 (δ_C 163.34, ²J_CH) and CH-6 (δ_C 95.45, ³J_CH) were used to characterize definitively the 1a structure. Other correlations revealed by HMBC are shown in Table I. The iodine derivative 1b was also characterized by same procedure, which revealed the data described in Table I.

However, the same reaction using the isoflavone (2) yielded different products when compared with those obtained of flavonol 1, by the presence of two additional methoxyl groups located at CH-2 and C-3 of the enone system (Figs. 1 and ³). The CG-LREIMS of the mixture containing the compounds 2a and 2b showed two peaks [T_R 19.0 min (58.14%) and T_R 25.3 (41.86%)] and the corresponding molecular ions peaks identified at m/z 486 (100%, 2a) and 612 (100%, 2b), as summarized in Figures 4 and 5. Besides these data, the analysis of ¹H and ¹³C NMR let us to identify the chemical shifts represented by two close values (Table I), suggesting similar structures compatible with the presence of these two enantiomeric pairs (2a and 2b). The detailed mass spectral data analysis and the NOEDIFF experiments justified both similar structures with two additional methoxyl groups with one and two incorporated iodine in 2a and 2b, respectively. These structures and locations of those groups were recognized by analysis of additional peaks in the mass spectra m/z (%): 2a: 486 (100, C₁₉H₁₉O₇I); 456 (40, M-30), 194 (90), 179 (40), 151 (25) Figure 4; 2b: m/z 612 (100, C₁₉H₁₈O₇I₂), 582 (10), 552 (10), 418 (9), 194 (90), 151 (40); been 194 = H₃COCH=COCH₃(C₆H₄-OCH₃) by RDA, Figure 5. The data of ¹H and ¹³C NMR ({¹H} and DEPT) 1D- and 2D (¹Hx¹H-COSY, HMQC and HMBC) spectra analysis are described in Table I. The NOEDIFF experiments were also used to confirm these structures, contributing to complete ¹H and ¹³C NMR assignments (Table I) and to identify the relative configuration of C-2 and C-3. The irradiations at δ_H 3.63 (H₃CO-2, 2a and 2b) yield NOE at H-2 [5.22 (2a) and δ_H 5.18 (2b)], irradiation at 3.19 (H₃CO-3, 2b) yield NOE at H-2 (δ_H 5.18, 2b), HO-5 (δ_H 12.17) and at H-2',6'(δ_H 7.42); irradiation at δ_H 3.86 (H₃CO-4" of 2a) yielded NOE at δ_H 6.98 (H-3',5') and at δ_H 3.95 (H₃CO-7 of 2a) yielded NOE at 6.23 (H-6); irradiation at δ_H 3.21 (H₃CO-3, 2a) yield NOE at δ_H 5.22 (H-2), δ_H 7.39 (H-2',6') and HO-5 (δ_H 12.81); no signal was observed in the spectrum obtained from irradiation at δ_H 3.97 (H₃CO-7 of 2b); on the other hand a doublet at δ_H 6.96 (H-3',5') was observed in the spectrum obtained by irradiation at δ_H 3.87 (H₃CO-4' of 2b). Thus the NOE observed at H-2 and H-3 with irradiation at the H₃CO-2 was used to justify the trans correlation relationship between methoxyl groups at 2 and 3 positions, according to the proposed mechanism summarized in ^{Figure 3}. The detailed analysis of ¹H and ¹³C NMR (1D and 2D) spectra let us to make the complete data assignments of these new derivatives (Table I).

In this work we rationalize trans stereoselectivity observed at the α, β-enone system of 2 as a well established addition of methanol to an intermediate halonium species (Bateman et al. 1983), followed by alkoxide substitution of the resulting β-alkoxy-(or β-hydroxy-)-α-halo derivative intermediate (Bird et al. 1983). Presumably, solvolytic conditions in methanol drives the incipient benzylic carbenium formed to be anchimeric assisted by a methoxy group (Smith et al. 1965), thus explaining the trans selectivity observed in the products. The reactions performed in the present work are of exploratory nature, carried out to ascertain experimental conditions and to ensure the observed products.

As far as we know, this is the first work that describes the incorporation of nuclear iodine atoms in flavonoids 1 and 2 along with the stereoselective trans incorporation of two methoxyl groups at the 2 and 3 positions of compound 2.

ACKNOWLEDGMENTS

The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Programa de Apoio Científico e Tecnológico/Financiadora de Estudos e Projetos (PADCT/FINEP) for fellowships and financial support. We also thank Centro Nordestino de Aplicação e Uso da Ressonância Magnética Nuclear (CENAUREMN) for the 500 MHz NMR spectra and Instituto de Química - Universidade Federal do Rio de Janeiro (IQ-UFRJ) for the 300 MHz NMR spectra.

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

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*

Member Academia Brasileira de Ciências

Publication Dates

• Publication in this collection
  19 Feb 2009
• Date of issue
  Mar 2009

History

• Accepted
  04 Nov 2008
• Received
  19 Nov 2007