Alkaloids from Annona dioica

Abstracts

From the ethanolic extract of the wood of Annona dioica were isolated the known 1-aza-4-methylanthraquinone, lasiodiplodin, liriodenine, a mixture of 1-aza-5,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene and 1-aza-8,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (geovanine) and the new alkaloid 1,2-methylenedioxy-6alpha,7-dehydroaporphine-4(S )-(4-hydroxy-3,5-dimethoxyphenyl)-3,4-dihydro-2(1H)-pyridinone. The structures of these natural products were elucidated on the basis of their spectral data, including NOE experiments and homonuclear ¹H-¹H-COSY e heteronuclear ¹H-13C-COSY-nJ CH (HMQC, n=1 and HMBC, n=2 and 3) 2D-shift-correlated NMR spectra.

Annona dioica; Annonaceae; quinoline alkaloids; lignoaporphine; lasiodiplodin

Do extrato etanólico da madeira de Annona dióica foram isoladas as conhecidas 1-aza-4-metilantraquinona, lasiodiplodina, liriodenina, uma mistura de 1-aza-5,9,10-trimetoxi-4-metil-2-oxo-1,2-diidroantraceno e 1-aza-8,9,10-trimetoxi-4-metil-2-oxo-1,2-diidroantraceno (geovanina) e o novo alcalóide 1,2-metilenodioxi-6alfa,7-desidroaporfina-4(S)-(4-hidroxi-3,5-dimetoxifenil)-3,4-diidro -2(1H)-piridinona. As estruturas destes produtos naturais foram elucidadas com base em seus dados espectrais, inclusive experiências de NOE e espectros de RMN 2D de correlação homonuclear ¹H-¹H-COSY e heteronuclear ¹H-13C-COSY-nJ CH (HMQC, n=1 e HMBC, n=2 e 3).

ARTICLE

Alkaloids from Annona dioica

Paulo R. D. dos Santos^I; Anselmo A. Morais^I; Raimundo Braz-Filho^II

^ICurso de Pós-graduação em Química Orgânica, Departamento de Química, ICE, UFRRJ, 23851-970 Seropédica - RJ, Brazil

^IISetor de Química de Produtos Naturais, LCQUI, CCT, Universidade Estadual do Norte Fluminense, 28015-620 Campos - RJ, Brazil

ABSTRACT

From the ethanolic extract of the wood of Annona dioica were isolated the known 1-aza-4-methylanthraquinone, lasiodiplodin, liriodenine, a mixture of 1-aza-5,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene and 1-aza-8,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (geovanine) and the new alkaloid 1,2-methylenedioxy-6a,7-dehydroaporphine-4(S )-(4-hydroxy-3,5-dimethoxyphenyl)-3,4-dihydro-2(1H)-pyridinone. The structures of these natural products were elucidated on the basis of their spectral data, including NOE experiments and homonuclear ¹H-¹H-COSY e heteronuclear ¹H-¹³C-COSY-ⁿJ_CH (HMQC, n=1 and HMBC, n=2 and 3) 2D-shift-correlated NMR spectra.

Keywords: Annona dioica, Annonaceae, quinoline alkaloids, lignoaporphine, lasiodiplodin

RESUMO

Do extrato etanólico da madeira de Annona dióica foram isoladas as conhecidas 1-aza-4-metilantraquinona, lasiodiplodina, liriodenina, uma mistura de 1-aza-5,9,10-trimetoxi-4-metil-2-oxo-1,2-diidroantraceno e 1-aza-8,9,10-trimetoxi-4-metil-2-oxo-1,2-diidroantraceno (geovanina) e o novo alcalóide 1,2-metilenodioxi-6a,7-desidroaporfina-4(S)-(4-hidroxi-3,5-dimetoxifenil)-3,4-diidro -2(1H)-piridinona. As estruturas destes produtos naturais foram elucidadas com base em seus dados espectrais, inclusive experiências de NOE e espectros de RMN 2D de correlação homonuclear ¹H-¹H-COSY e heteronuclear ¹H-¹³C-COSY-ⁿJ_CH (HMQC, n=1 e HMBC, n=2 e 3).

Introduction

Annona dioica, Annonaceae, is a shrub distributed throughout the States of São Paulo, Minas Gerais, Paraná and Mato Grosso, Brazil, commonly called "ceraticum (do campo, or grande)", "arixicum" and "ariticum". The fruits and leaves are used against rheumatism and the seeds to heal diarrhea.¹

This paper reports the isolation of the known 1-aza-4-methylanthraquinone (1), lasiodiplodin (2), liriodenine (3), along with a mixture of 1-aza-8,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (4, geovanine) and 1-aza-5,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (5), and the new alkaloid 1,2-methylenedioxy-6a,7-dehydroaporphine-4(S )-(4-hydroxy-3,5-dimethoxyphenyl)-3,4-dihydro-2(1H)-pyridinone (6). The structures were established by spectral analysis, mainly ¹H and ¹³C NMR including homonuclear 2D ¹H-¹H-COSY and heteronuclear 2D ¹H-¹³C-COSY-ⁿJ_CH (HMQC, n=1 and HMBC, n=2 and 3) and NOE experiments.

To the best of our knowledge, the compound 6 is hitherto unreported in the literature.

Results and Discussion

Comparative analysis of HBBD- and DEPT-¹³C NMR spectra of each natural product (1 to 6) was used to identify signals corresponding to quaternary, methine, methylene and methyl carbon atoms. Compound 1 was previously isolated from Cleistopholis patens,^2,32 from Lasiodiplodia theobromae⁴ and Euphorbia splendens,⁵3 from Atherosperma moschatum, Liriodendron tulipifera,⁶Fusea longifolia, Siparuna guianensis,⁷Thalictrum sessile⁸ and many other plants species, and 4 (geovanine) from Annona ambotay.⁹ The structural identification of these compounds was based on their spectral data, including homonuclear 2D ¹H-¹H-COSY and heteronuclear 2D ¹³C-¹H-COSY-ⁿJ_CH (n=1; n=2 and 3, COLOC) experiments and differential NOE data, together with comparison with literature values of ¹H and ¹³C NMR (vide supra).

The mixture of 4 and 5 showed IR absorption for conjugated carbonyl function (n_max 1650 cm^-1) and aromatic ring (n_max 1560 and 1520 cm^-1). The multiplicity of each carbon signal of the two components 4 and 5 was deduced by comparative analysis of the HBBD- and DEPT-¹³C NMR spectra (Table 1). This analysis in combination with GC-EIMS {Rt=2.41min (40%): m/z 299 ([M]⁺ of 5, 65%; Rt=3.08 min (60%): m/z 299 ([M]⁺ of 4, 63%)} and 1D ¹H and 2D ¹H-¹H-COSY NMR spectra (Table 1) allowed the deduction of the same molecular formula C₁₇H₁₇NO₄ for 4 and 5. Comparison of ¹H NMR spectral data of 4 and of geovanine (isolated from Annona ambotay⁹) was used to characterize it as the same compound (Table 1) and consequently to establish the structure of 4 as 1-aza-8,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene. This structure was confirmed by NOE difference experiments performed with irradiation at MeO-10 (d_H 3.90) which resulted in signal enhancements at d_H 2.75 (Me-4) and 7.75 (H-5) and irradiation at MeO-8 (d_H 4.02) which showed NOE at d_H 6.88 (H-7). The remaining signals observed in the ¹H (d_H 7.82, t, J 8.2 Hz, H-7, position conjugated with carbonyl group; partial superimposition with signal at 7.75 of the H-5 of 4), 6.59 (s, H-3), 2.65 (s, Me-4), 4.04 (s, MeO)] and ¹³C (d_C 63.79, MeO)] NMR and GC/MS (vide supra) spectra were speculatively used to propose the structure 5 (1-aza-5,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydro anthracene) for the other component present in the mixture, probably a new alkaloid.

The lignoaporphine alkaloid 6 showed IR absorption bands for a carbonyl group (n_max 1660 cm^-1) and for aromatic ring (n_max 1630, 1610, 1520 and 1500 cm^-1). The number of hydrogen atoms bound to each carbon atom was deduced by comparative analysis of the HBBD - (25 signals corresponding to 28 carbon atoms) and DEPT-¹³CNMR (Table 2) in combination with ¹H NMR spectral data (1D and 2D ¹H-¹H-COSY) and low resolution mass spectrum (m/z 469 [M]⁺, 100%, C₂₈H₂₃NO₆), allowing to establish the formula (C)₁₃(C=O)(CH)₈(CH₂ )₃(OCH₂O) (MeO)₂(OH)= C₂₈H₂₃O₆ (455 daltons) + N = C₂₈H₂₃NO₆ (m/z 469, [M]⁺). This molecular formula is compatible with an aporphine skeleton (C₁₆H₉N) sustaining one methylenedioxy group and one 4'-hydroxy-3',5'-dimethoxy-7',8'-dihydrocinnamoyl moiety in a lactam ring. In fact, the presence of this cinnamoyl system was evidenced by the chemical shifts of 2H-8' [d_H 3.20 (dd, J 14.5; 6.2 Hz); 3.07 (br d, J 14.5 Hz)], H-7' [d_H 4.80 (br d, J 6.2 Hz)], 2H-2',6' [d_H 6.38 (s)] and 2MeO-3',5' [d_H 3.74 (s)] in addition to the chemical shifts of C-1' (d_C 131.77), 2CH-2',6' (d_C 103.82), 2C-3',5' (d_C 147.35), C-4' (d_C 133.96), CH-7' (d_C 37.96), CH₂-8' (d_C 39.90) and C-9' (d_C 167.65). The location of the cinnamoyl unit, involving carbon C-7 and the nitrogen atom, was defined by heteronuclear 2D ¹H-¹³C long-range couplings (²J_CH and ³J_CH) between: C-7 (d_C 115.85) and H-7' (d_H 4.80, ²J_CH), 2H-8' (d_H 3.20; 3.07, ³J_CH) and H-8 (d_H 7.85, ³J_CH); C-6a (d_C 132.22) and H-5 (d_H 5.29, ³J_CH) and H-7' (d_H 4.80, ³J_CH); C-9' (d_C 167.65) and H-5 (d_H 5.29, ³J_CH), H-7' (d_H 4.80, ³J_CH) and 2H-8' (d_H 3.20 and 3.07, ²J_CH). The remaining signals observed in the ¹H and ¹³C NMR spectra (Table 2) were used to define the 2,3-methylenedioxyaporphine [(C)₉(CH)₅(CH₂)₂(OCH ₂O)] unit, being the homo- and heteronuclear correlations obtained from 2D ¹H-¹H-COSY and ¹H-¹³C-COSY-ⁿJ_CH (n=1, HMQC; n=2 and 3, HMBC) spectra summarized in Table 2. The 2D shift-correlated spectra were also used to complete ¹H and ¹³C chemical shift assignments of 6. The following cross-peaks observed in the NOESY spectrum were also utilized for additional confirmation of the structure proposed to 6: a) NOE between H-8 (d_H 7.85) and H-7' (d_H 4.80) and 2H-2',6' (d_H 6.38); b) NOE between H-7'(d_H 4.80) and 2H-2',6' (d_H 6.38); c) NOE between 2H-2',6' (d_H 6.38) and 2MeO-3',5' (d_H 3.74); d) NOE between H-3 (d_H 7.13) and pseudoequatorial H-4 (d_H 3.19).

Additional peaks observed in the low resolution EIMS at m/z 316 (96%), 314 (22%) and 286 (26%) attributed to fragments 6a, 6b and 6c, respectively, are also consistent with structure 6.

Thus, the structure of the new lignoaporphine alkaloid 6 was established as 1,2-methylenedioxy-6a,7-dehydroaporphine-4-(4-hydroxy-3,5-dimethoxyphenyl)-3,4 -dihydro-2(1H)-pyridinone. The [a]_D of + 326^o (CHCl₃; c 2.50) indicated that it was not a racemic mixture, ruling out the possibility of 6 being an artifact. The assignment of the absolute stereochemistry at the chiral centre CH-7' of the 3-(4-hydroxy-3,5-dimethoxyphenyl)propanoyl moiety of 6 was postulated by comparison of its optical rotation [a]_D + 326^o (CHCl₃; c 2.50) with those reported for structures 7-10, which contain an asymmetric carbon with nearly identical substituents. In those compounds the S configuration gave an optical rotation about 200^o higher than the R configuration.¹⁰ Consequently, the [a]_D + 326^o (CHCl₃; c 2.50) is consistent with (S)-stereochemistry of the chiral carbon CH-7' present in the 3-(4-hydroxy-3,5-dimethoxyphenyl)propanoyl unit of 6.

Thus, the structure of new lignoaporphine alkaloid was established as 1,2-methylenedioxy-6a,7-dehydroaporphine-4(S )-(4-hydroxy-3,5-dimethoxyphenyl)-3,4-dihydro-2(1H)-pyridinone, IUPAC name 5-(4-hydroxy-3,5-methoxyphenyl)-(5S)-6,7,9,10-tetrahydro-5 H-benzo[g][1,3]dioxolo[4',5':4,5]benzo[de]pyrido[3,2,1- ij]quinolin-7-one (6).

Experimental

General experimental procedures

Mps are uncorr. NMR spectra were run on Bruker AC-200 and Advance 500 spectrometers in CDCl₃ or CD₃COCD₃ using TMS as internal standard or by reference to the solvent signal (CHCl₃ at d_H 7.24 or CD₂HCOCD₃ at d_H 2.08 and CDCl₃ at d_C 77.00 or CD₃COCD₃ at d_C 24.8 and 206.0). EIMS were obtained at 70 eV on a Hewlett Packard spectrometer model 5987. The IR spectra were obtained on a Perkin-Elmer FT-1500 spectrometer. Column chromatography was carried out with silica gel 0.063–0.2 mm and TLC was done employing silica gel Kieselgel 60 from Merck and spots were visualized by UV (l_max 259 and 360 nm) and exposure to I₂ vapour.

Plant material

A specimen of Annona dioica St. Hil. was collected in January 1990 at Serra da Moeda, Minas Gerais State, Brazil and identified by Professor José Badini of the Universidade Federal de Ouro Preto, Minas Gerais, where a voucher specimen is deposited.

Extraction and isolation of constituents

Dried and powdered wood (5.2 kg) was extracted successively with n-hexane and EtOH at room temp. and the solvent removed under vacuum to yield 81.79 g of residue. This residue was chromatographed on a silica gel column using CH₂Cl₂, CHCl₃, n-hexane-EtOAc (4:1, 3:2, 2:3 and 1:4), EtOAc and EtOAc-MeOH (4:1 and 1:1). Fractions 52-71, eluted with CH₂Cl₂, were rechromatographed on a silica gel column (70-230 mesh) using CHCl₃ and CHCl₃-MeOH (97:3) as eluents to furnish 15 fractions: fractions 6-10 yielded 1 (22 mg) and 2 (20 mg) after chromatography on a silica gel column using CHCl₃-MeOH (97:3). Preparative TLC (CHCl₃-MeOH, 97:3) of the fractions 98-115 (eluted with CHCl₃-MeOH 97:3) afforded 3 (15 mg) and 6 (83 mg). Fractions 135-229 (4.7 g, eluted with CHCl₃-MeOH 97:3) were chromatographed on a silica gel (100 g) column using CH₂Cl₂, CH₂Cl₂-CHCl₃ (1:1 and 1:4), CHCl₃, n-hexane-EtOAc (1:4) and MeOH to furnish 4+5 (18.8 mg) as amorphous yellow solid after treatment with acetone.

1-Aza-4-methylanthraquinone (1). Oil. Spectral data in agreement with literature values.^2,3

12-Hydroxy-14-methoxy-3-methyl-3,4,5,6,7,8,9,10 -octahydro-1H-benzo[c]oxa-cyclododecin-1-one (2, Lasidioplodin). Amorphous yellow solid, mp 183-186 ^oC; spectral data in agreement with lit. values.^4,5

8H-Benzo[g][1,3]dioxolo[4',5':4,5]benzo [de]quinolin-8-one (3, Liriodenine). Amorphous yellow solid, mp 284-286 ^oC; spectral data in agreement with lit. values.^6-8

Mixture of 1-Aza-8,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (4, geovanine) and 1-aza-5,9,10-trimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (5). Amorphous yellow solid, mp 189-191 ^oC; IR (KBr) n_max/cm^-1: 1650 (C=O), 1560 and 1520 (aromatic ring); GC Rt = 2.418 (5)/3.086 (4) min; EIMS (rel. int., 4/5) m/z: 299 ([M]⁺, 63/65), 284 (M-Me^., 100/100), 269 (M-CH₂O, 76/72), 268 (M-MeO, 7/7), 252 (M-CH₂O-OH, 9/9), 240 (M-MeO^.-C=O, 17/18); ¹H and ¹³C NMR of 4: Table 1.

1,2-methylenedioxy-6a,7-dehydroaporphine-4( S)-(4-hydroxy-3,5-dimethoxyphenyl)-3,4-dihydro-2(1 H)-pyridinone (6). Amorphous yellow solid, mp 211-213 ^oC; IR (KBr) n_max/cm^-1: 3600, 3500, 1660, 1630, 1610, 1520, 1500, 1260, 1050, 850, 720 cm^-1, ¹H and ¹³C NMR: Table 2.

Acknowledgements

This work was supported by a fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and grants from Financiadora de Estudos e Projetos (FINEP)/Programa de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT), Coordenação de Aperfeiçoamento de Pessoal Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). The authors also wish to thank Professor José Badini of the Universidade Federal de Ouro Preto, Minas Gerais, Brazil for the plant identification.

Received: April 5, 2002

Published on the web: March 24, 2003

* e-mail: [e-mail]

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Publication Dates

• Publication in this collection
  16 June 2003
• Date of issue
  May 2003

History

• Accepted
  24 Mar 2003
• Received
  05 Apr 2002