Spectral Assignments and Reference Data Received: 29 September 2008 Revised: 30 January 2009 Accepted: 1 February 2009 Published online in Wiley Interscience: 23 March 2009 (www.interscience.com) DOI 10.1002/mrc.2422 Unequivocal assignments of flavonoids from Tephrosia sp. (Fabaceae) A. M. C. Arriaga,a∗ J. Q. Lima,a J. N. Vasconcelos,a M. C. F. de Oliveira,a M. Andrade-Neto,a G. M. P. Santiago,a,b D. E. A. Uchoa,a G. T. Malcher,a J. Mafezolia and R. Braz-Filhoc 1 H and 13 C NMR chemical shifts of praecansone B, pongaflavone and dehydrorotenone isolated from Tephrosia egregia Sandw and obovatin from T. toxicaria Pers. were unambiguously assigned by 1D and 2D NMR experiments including 1 H, 1 H COSY, c 2009 John Wiley & Sons, Ltd. gHMQC and gHMBC, allowing the correction of literature assignments. Copyright
Keywords: NMR; 1 H NMR; 13 C NMR; 1D/2D NMR; praecansone B; pongaflavone; obovatin; dehydrorotenone; Tephrosia; Fabaceae Introduction Tephrosia (Fabaceae) is a large perennial genus distributed in the tropical and sub-tropical regions of the world.[1] Most Tephrosia species possess insecticidal, fish-poisoning and medicinal proprieties and the phytochemical investigation of this genus provided steroids, chalcones, flavanones, flavones, pterocarpanes and rotenoids as secondary metabolites.[2 – 4] In the course of our study of the genus Tephrosia, we have characterized the essential oil from Tephrosia egregia Sandw[5] and Tephrosia toxicaria Pers.[6] ; the later presented antioxidant and insecticidal (third instar Aedes aegypti larvae) activities.[7] As part of our ongoing project, herein we report the complete 1 H and unequivocal 13 C NMR chemical shift assignments of praecansone B (1), pongaflavone (2) and dehydrorotenone (4) isolated from T. egregia, and obovatin (3) isolated from T. toxicaria, based on 1D and 2D NMR techniques. Although these compounds have been reported previously in literature,[8 – 12] their NMR data are rather old or incomplete. Therefore, for reference purpose it is useful to report the NMR data of these compounds based on modern 2D NMR measurements. Additionally, in order to provide a set of data that might serve as models for the assignments of similar compounds in further research, the 13 C NMR chemical shifts assignments reported in the literature[8 – 12] for all compounds were corrected. reported by Tarus et al.[8] in 2002. However, C-2/C-6, C-3/C-5, C-2′ , C-4′ , C-3′′ and C-4′′ were incorrectly assigned,[8] which are now corrected. The gHMBC spectrum analysis allowed the unequivocal assignment of C-2/C-6 and C-3/C-5 chemical shifts by the correlation peaks between the hydrogens at δH 7.94 (H-2/H-6) with the carbons at δC 132.2 (C-4) and δC 182.2 (C-7). The correct assignment of C-2′ and C-4′ was done through the correlation of the hydrogens at δH 3.80 (CH3 O-2′ ) and δH 6.54 (H-4′′ ) with the carbon at δC 158.6 (C-2′ ); and δH 6.26 (H-5′ ) and δH 6.54 (H-4′′ ) with the carbon at δC 155.4 (C-4′ ). The assignments of C-3′′ and C-4′′ were obtained by the correlation between the methyl signals at δH 1.46 (CH3 -2′′ ) from the gem-dimethylchromene moiety with the carbon at δC 127.9 (C-3′′ ). These assignments were corroborated by the correlation of the signal at δH 6.54 (H-3′′ ) with carbons at δC 77.4 (C-2′′ ) and δC 28.1 (CH3 -2′′ ). Additionally, the signal of H-4′′ at δH 5.55 showed correlations with carbons at δC 77.4 (C-2′′ ), δC 155.4 (C-4′ ) and δC 158.6 (C-2′ ). Compound 2 was obtained as a yellow amorphous solid, mp 199.3–201.4 ◦ C. Its molecular formula, C21 H18 O4 , was suggested by a combination of EIMS ([M]+ · m/z 336) and NMR data. Analysis of all spectral data (1 H and 13 C NMR, and MS; Table 2) suggested that 2 has some similarities with 1; the main differences were that, 2 had only one methoxyl group at δH 3.95 (3H, s), two singlet methine hydrogens (δH 6.33, s, H-6 and δH 6.67, s, H3), and lacked a hydroxyl hydrogen. Moreover, the 1 H NMR spectrum of 2 showed the presence of the same signals for Results and Discussion Magn. Reson. Chem. 2009, 47, 537–540 ∗ Correspondence to: A. M. C. Arriaga, Curso de Pós-graduação em Química, Departamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, Cx. Postal 12200, Fortaleza-CE, 60451-970, Brazil. E-mail: angelamcarriaga@yahoo.com.br. a Curso de Pós-graduação em Química, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Cx Postal 12200, CEP 60451-970, Fortaleza - CE, Brazil b Departamento de Farmácia, Universidade Federal do Ceará, Rua Capitão Francisco Pedro 1210, CEP 60430-370, Fortaleza - CE, Brazil c Pesquisador Visitante 1-CNPq/Programa de Pós-graduação em Química (PPGQ), Universidade Federal do Ceará, Fortaleza – CE, Brazil c 2009 John Wiley & Sons, Ltd. Copyright
537 Praecansone B, 1, was obtained as yellow gummy material. Its molecular formula, C22 H22 O5 , was suggested on the basis of its EIMS ([M]+ · m/z 366) and comparative analysis of its 1 H, HBBD and DEPT-13 C NMR spectral data (Table 1). Analysis of 1 H and 13 C 1D/2D NMR data (Table 1) allowed the identification of a gemdimethylchromene moiety, a hydroxyl group strongly chelated to a carbonyl group, an unsubstituted benzene ring, two methoxyl groups and a hydroxylated vinyl group. This led to complete assignment of the whole β-hydroxy-prenilated chalcone structure of Praecansone B (1) (Fig 1). This compound has been previously identified from Tephrosia aequilata and its carbon signals were A. M. C. Arriaga et al. Table 1. 13 C and 1 H NMR data for praecansone B (1) 1H C 1 2, 6 3, 5 4 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 2′′ 3′′ 4′′ (CH3 )2 -2′′ CH3 O-2′ CH3 O-6′ HO-7 a b δ 13 Ca × 13 C – HMQC [1 J (C, H)] (Ref. [8]) 134.0 128.5 127.0 132.1 182.0 100.5 188.0 108.0 155.0 114.0 158.2 96.2 156.2 76.4 116.5 127.7 28.0 56.1 63.2 – δ 13 C a 135.3 127.2 128.7 132.2 182.2 100.7 188.2 108.3 158.6 114.5 155.4 96.4 156.6 77.4 127.9 116.7 28.1 56.2 63.3 – δ Hb HMBC – – 7.94 (d, 7.3) 4; 7 7.45 (t, 7.3; 7.75) – 7.55 (t, 7.75) – – – 6.50 (s) 7; 9 – – – – – – – – – – 6.26 (s) 4′ ; 6′ ; 1′ ; 3′ – – – – 5.55 (d, 10.0) 2′′ ; (CH3 )2 -2′′ 6.54 (d, 10.0) 4′ ; 2′ ; 2′′ 1.46 (s) 2′′ ; 3′′ 3.80 (s) 2′ 6′ 16.29 (s) – CDCl3 , 125 MHz. CDCl3 , 500 MHz. the dimethylchromene moiety and B unsubstitued benzene ring as of 1. These observations pointed out the presence of a basic flavone skeleton in 2. The 2D shift-correlated NMR (1 H-1 H-COSY, gHMQC and gHMBC) experiments were used to establish the structure of 2 as pongaflavone (Fig. 1) and correct the assignment of C-3′′ and C-4′′ chemical shifts previously reported.[9,10] The unequivocal assignment of these two carbons was done by the gHMBC correlation of the hydrogens at δH 1.50 (CH3 -2′′ ) with the carbon at δC 127.7 (C-3′′ ). All 1 H and 13 C 1D/2D NMR data of 2 are shown in Table 2. The structure of compound 3 is very similar to that of 2 and it was obtained as a yellow amorphous solid, mp 119.8–121.2 ◦ C. Its molecular formula, C20 H18 O4 , was deduced by a combination of EIMS ([M]+ · m/z 322) and 13 C NMR (HBBD and DEPT, 135◦ pulse sequence) data. The basic flavanone skeleton was present in 3 as indicated in the 1 H NMR spectrum by three characteristic doublets of doublets (δH 5.44, dd, J = 13.0 and 3.0 Hz, H-2 and δH 3.06, dd, J = 17.1 and 13.0 Hz, H-3ax and δH 2.85, dd, J = 17.1 and 3.0 Hz, H-3eq ). Additionally, as observed in 2, compound 3 showed characteristic signals of one non-coupled aromatic hydrogen and a dimethylchromene, both in the ring A and B unsubstituent benzene ring. Further analysis of its 2D shift-correlated NMR (1 H-1 H-COSY, gHMQC and gHMBC) spectra led to the complete assignment of the whole structure 3, identified as obovatin[11] (Table 2). However, C-5, C-7 and C-8a were incorrectly assigned in the literature.[11] The correlation of the hydrogen at δH 12.09 (OH) with carbons at δC 164.0 (C-5), δC 97.8 (C-6) and δC 103.1 (C-4a), observed in the gHMBC spectrum, allowed the correct assignment of C-5. The unequivocal assignments of C-7 and C-8a were obtained by the gHMBC correlation of the signal at δH 5.44 (H-2) with the carbon at δC 156.9 (C-8a) and the signal at δH 6.56 (H-4′′ ) with the carbons at δC 156.9 (C-8a) and δC 162.5 (C-7). Compound 4 was obtained as yellow crystals, mp 134.6–136.5 ◦ C. The molecular formula was established as C23 H20 O6 from EIMS ([M]+· m/z 392) and 13 C NMR (HBBD and DEPT-13 C NMR, 135◦ pulse sequence) data. Analysis of the 1 H NMR spectrum revealed characteristic signals of 1-(methylethenyl)dihydrofuran ring with diastereotropic hydrogens at C-4′ (δH 3.54, 1H, dd, J = 15.8 and 10.0 Hz and δH 3.20, 1H, dd, J = 15.8 and 7.9 Hz), 5′ (δH 5.42, 1H, t, J = 8.8 Hz), 7′ (δH 1.82, 3H, s) and 8′ (δH 5.14 and 4.99, 2H, s). Additionally, signals of four aromatic hydrogens at δH 8.46 (1H, s, H-1), δH 8.14 (1H, d, J = 8.6 Hz, H-11), δH 6.93 (1H, d, J = 8.6 Hz, H-10) and δH 6.56 (s, 1H, H-4), and two methoxyl groups (δH 3.96, 3H,s and δH 3.88, 3H,s) were observed. 538 Figure 1. Chemical structures of compounds 1–4. www.interscience.wiley.com/journal/mrc c 2009 John Wiley & Sons, Ltd. Copyright
Magn. Reson. Chem. 2009, 47, 537–540 Spectral Assignments and Reference Data Table 2. 13 C and 1 H NMR data for pongaflavone (2) and obovatin (3) Compound 2 HMQC [1 J Compound 3 HMQC [1 J (C, H)] (C, H)] C δ 13 Ca (Ref. [9,10)] δ 13 Ca δ Hb HMBC δ 13 Ca (Ref. [11]) δ 13 Ca δ Hb HMBC 2 160.8 160.8 – – 78.9 79.2 1′ ; 4; 8a; 2′ 3 109.0 109.0 6.67 (s) 2; 4; 1′ 43.1 43.5 4 5 6 7 8 4a 8a 1′ 2′ , 6′ 3′ , 5′ 4′ 2′ ’ 3′′ 177.6 154.0 96.7 160.2 102.8 109.0 158.0 131.9 125.9 129.0 131.2 78.1 115.3 177.8 154.1 96.8 160.4 102.8 109.0 158.1 131.9 126.0 129.1 131.3 78.2 127.7 – – 7; 8 – – – – – 2′ ; 4′ ; 2 2′ ; 1′ 3′ ; 2′ – 2′′ ; 8; (CH3 )2 -2′′ 196.0 157.0 97.5 164.0 102.0 103.0 162.0 139.0 125.9 128.7 128.6 78.0 126.3 195.8 164.0 97.8 162.5 102.1 103.1 156.9 138.7 126.2 129.0 129.0 78.3 126.7 – – 7; 8; 4a – – – – – 2 1′ – – 2′′ ; 8; (CH3 )2 -2′′ 4′′ 127.6 115.4 8; 2′′ ; 8a 115.4 115.8 6.56 (d, 10.0) 8; 7; 8a; 2′′ 56.5 28.3 56.6 28.4 – – 6.33 (s) – – – – – 7.86 (m) 7.50 (m) 7.50 (m) – 5.62 (d, 10.0) 6.85 (d, 10.0) 3.95 (s) 1.50 (s) 5.44 (dd, 13.0; 3.0) 3.06 (dd, 17.1; 13.0-Hax ) 2.85 (dd, 17.; 3.0-Heq ) – – 6.02 (s) – – – – – – 7.40–7.48 (m) – – 5.48 (d, 10.0) 5 2′′ ; 3′′ – – – – 28.4 28.7 – s1.44 (s) 1.46 (s) 12.09 (s) – 2′′ ; 3′′ – – 28.1 28.4 – CH3 O-5 (CH3 )2 -2′′ –OH a b 4; 2; 4a; 1′ 5; 4a; 6 CDCl3 , 125 MHz. CDCl3 , 500 MHz. This analysis suggested that 4 was a rotenone derivative, considering that rotenoids were previously isolated from Tephrosia sp.[13] The lack of hydrogen signals at 6a and 12a positions in the rotenone skeleton, suggested for compound 4 the structure of 6a, 12a-dehydrorotenone. The application of 1D and 2D techniques was used to establish the unambiguous assignments of the 1 H NMR and 13 C NMR for 4 (Table 3). The assignment of C-1a, C-6a, C-7a, C-8 and C-11a of 4 is strongly supported by gHMBC spectrum, which showed connectivity between the signals at δH 6.56 (H-4) and δC 110.8 (C-1a) and δC 144.3 (C-2); between δH 5.01 (H-6) and δC 156.2 (C-6a), δC 111.9 (C-12a) and δC 146.5 (C-4a); between δH 8.14 (H-11) and δC 152.5 (C-7a), δC 165.0 (C-9) and δC 174.6 (C-12); between δH 6.93 (H-10) and δC 113.1 (C-8) and δC 119.2 (C-11a). The position of C-8 was confirmed by the correlation of the signals at δH 3.54 and δH 3.20 (H-4′ ) with δC 113.1 (C-8). The 13 C NMR assignment of these carbons differed from those previously reported in the literature[13] for compound 4 and was corrected. Experimental Plant material Magn. Reson. Chem. 2009, 47, 537–540 Air-dried and powdered leaves of T. egregia (1100.0 g), obtained after essential oil extraction, were extracted with ethyl acetate for 72 h at room temperature, which provided 59.0 g of a dark green material. An aliquot of the extract (21.0 g) was submitted to column chromatography on silica gel using a gradient mixture of hexane, dichloromethane and ethyl acetate. The resulting fractions were combined into ten fractions. The fraction (650.0 mg) eluted with hexane-dichloromethane (1 : 1) and that eluted with ethyl acetate (322.2 mg) was rechromatographed over silica gel column to afford compounds 1 (32.0 mg) and 2 (110.0 mg) after elution with benzene and isocratic mixture of benzene-ethyl acetate (85 : 15), respectively. Air-dried and powdered roots of T. egregia were exhaustively extracted with methanol to afford a crude extract (5.3 g). This extract was chromatographed on silica gel using a gradient mixture of hexane, dichloromethane and ethyl acetate. The fraction eluted with dichloromethane was submitted to successive column chromatography on silica gel, and provided compound 4 (20.1 mg). Air-dried and powdered roots (109.0 g) of T. toxicaria were exhaustively extracted with ethanol at room temperature to afford c 2009 John Wiley & Sons, Ltd. Copyright
www.interscience.wiley.com/journal/mrc 539 T.egregia and T.toxicaria were collected in Flexeiras and Guaraciaba do Norte, respectively, Ceará State, northeast region of Brazil. Both specimens were identified by Dr. Edson de Paula Nunes, a plant taxonomist at Departamento de Biologia, Universidade Federal do Ceará, Brazil and their vouchers (#30389 and #32139) were deposited at the Herbarium Prisco Bezerra (EAC), Universidade Federal do Ceará, Brazil. Extraction and isolation A. M. C. Arriaga et al. Table 3. 13 C and 1 H NMR data for dehydrorotenone (4) 1H C δ 13 Ca × 13 C – HMQC [1 J (C, H)] (Ref. [12]) δ 13 Ca 1 1a 2 3 4 4a 6 6a 7a 8 9 10 11 11a 12 12a 4′ 108.5 118.8 143.9 148.3 100.3 146.1 64.7 152.1 155.9 110.5 164.7 110.0 127.7 111.5 174.1 112.9 31.4 110.2 110.8 144.3 149.1 100.6 146.5 65.1 156.2 152.5 113.1 165.0 109.0 128.1 119.2 174.6 111.9 31.6 5′ 6′ 7′ 8′ 87.8 142.7 17.0 112.8 88.1 143.0 17.2 113.1 56.2 55.8 56.5 56.1 CH3 O-2 CH3 O-3 a b δ Hb HMBC 8.46 (s) 12a; 4a; 3 – – – – – – 6.56 (s) 1a; 2 – – 5.01 (s) 6a; 12a; 4a – – – – – – – – 6.93 (d, 8.6) 8; 11a 8.14 (d, 8.6) 7a; 9; 12 – – – – – – 3.54 (dd, 15.8; 10.0) 8; 6′ ; 9; 5′ 3.20 (dd, 15.8; 7.9) 5.42 (t, 8.8) 7′ ; 8′ – – 1.82 (s) 6′ ; 5′ ; 8′ 5.14 (s) 7′ ; 5′ 4.99 (s) 7′ ; 5′ 3.96 (s) 2 3.88 (s) 3 CDCl3 , 125 MHz. CDCl3 , 500 MHz. a crude extract (16.0 g). Column chromatography of this extract on silica gel by hexane, chloroform, ethyl acetate and methanol provided four fractions. An aliquot of the second fraction (3.0 g), eluted with chloroform, was purified by flash chromatography using hexane-ethyl acetate-methanol (89 : 10 : 1), and provided compound 3 (57.7 mg). NMR spectra All experiments were performed on a Bruker DRX-500 spectrometer equipped with a 5 mm inverse detection z-gradient probe. 1 H (500.13 MHz) and 13 C (125.77 MHz) NMR spectra were measured at 27 ◦ C using CDCl3 . Chemical shifts are given on the δ scale and were referenced to residual CHCl3 (δH 7.27 and δC 77.23). The 1D and 2D 1 H and 13 C NMR spectra were performed by standard BRUKER’S pulse programs [zg30 (1 H), zgpg(13 C-BBHD), dept 135(13 C-DEPT 135◦ ), cosygpqf (1 H,1 H-COSY), hsqcgpph (1 H,13 C-HSQC), hmbcgplpndqf (1 H,13 C-HMBC)[14 – 16] ] using a 5 mm DUAL (13 C/1 H) probe for 13 C direct detection (13 C-BBHD and 13 CDEPT 135◦ ) and a 5 mm multinuclear inverse probe, with gradients coils in Z-axis, for 13 C inverse detection (1 H,13 C-HSQC and 1 H,13 CHMBC). Of all, 64 K data points, with a spectral width of 12 and 32 KHz, were collected for 1 H and 13 C 1D spectra, respectively. Inverse detected 2D heteronuclear correlation spectra were collected with 2048 × 256 matrix data points for 1 H,13 C-HSQC and 4096 × 256 matrix data points for 1 H,13 C-HMBC, and a spectral width of 5 KHz in F2 and 28 KHz in F1 for both experiments. Data processing was performed with 1 K × 256 matrix data points, with forward linear prediction in F1 (80 coefficients). Acknowledgements The Contract/grant sponsors for this article are: Fundação Cearense de Apoio a Pesquisa (FUNCAP), Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq/Pronex), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). References [1] B. Sinha, A. A. Natu, D. D. Nanavati, Phytochemistry 1982, 21, 1468. [2] H. M. A. Al-Hazimi, N. A. Al-Jaber, M. Rafiq, H. Siddiqui, J.SaudiChem. Soc. 2006, 9, 597. [3] M. Saleem, S. Ahmed, A. Alam, S. Sultana, Pharm. Res. 2001, 43, 135. [4] L. Crombie, D. A. Whiting, Phytochemistry 1998, 38, 1479. [5] A. M. C. Arriaga, F. E. A. Magalhães, E. M. A. Feitosa, G. T. Malcher, M. Andrade-Neto, R. F. Nascimento, J. Essent. Oil Res. 2005, 17, 451. [6] W. H. F. Ribeiro, A. M. C. Arriaga, M. Andrade-Neto, J. N. Vasconcelos, G. M. P. Santiago, R. F. Nascimento, J. Essent. Oil Res. 2006, 18, 95. [7] J. N. Vasconcelos, J. Q. Lima, T. L. G. Lemos, M. C. F. Oliveira, M. M. B. Almeida, M. Andrade-Neto, A. M. C. Arriaga, G. M. P. Santiago, J. Mafezoli, R. Braz-Filho, Quim. Nova 2009, In Press. [8] P. K. Tarus, A. K. Machocho, C. C. Lang’at-Thoruwa, S. Chhabra, Phytochemistry 2002, 60, 375. [9] P. G. Waterman, E. N. Mahmoud, Phytochemistry 1985, 24, 571. [10] M. A. Gissaub, S. A. Tarafdar, Indian J. Chem., Sect. B 1998, 37, 540. [11] C. C. Andrei, D. T. Ferreira, M. Faccione, L. A. B. Moraes, M. G. Carvalho, R. Braz-Filho, Phytochemistry 2000, 55, 799. [12] N. Fang, J. E. Casida, J. Org. Chem. 1997, 62, 350. [13] C. C. Andrei, P. C. Vieira, J. B. Fernandes, M. F. G. F. Da Silva, E. R. Fo, Phytochemistry 1997, 46, 1081. [14] S. Braun, H. O. Kalinowski, S. Berger, 150 and More Basic NMR experiments. A practical Course (2nd edn), Wiley-VHC: New York, 1998. [15] H. Günther, NMR Spectroscopy: Basic Principles, Concepts and Applications in Chemistry (2nd edn), John Wiley and Sons: New York, 1995. [16] T. D. K. Claridge, in In High-Resolution NMR Techniques in Organic Chemistry. Tetrahedron Organic Chemistry Series (Eds: J. E. Baldwin, R. M. Willians), Pergamon Press: Oxford, 1999. 540 www.interscience.wiley.com/journal/mrc c 2009 John Wiley & Sons, Ltd. Copyright
Magn. Reson. Chem. 2009, 47, 537–540