A new indole alkaloid isolated from Tabernaemontana hystrix steud (Apocynaceae)

Abstracts

A new alkaloid, named hystrixnine (1), and five known indole alkaloids, ibogamine (2), olivacine (3), affinine (4), affinisine (5) and Nb-methylaffinisine (6), were isolated from the root bark of Tabernaemontana hystrix. The known triterpenes alpha-amyrin acetate, beta-amyrin acetate and lupeol acetate were also identified. The structures of the compounds were elucidated based on spectroscopic studies.

Tabernaemontana hystrix; Apocynaceae; indole alkaloids; triterpenes

Um novo alcalóide, denominado histrixnina (1), e cinco alcalóides indólicos conhecidos, ibogamina (2), olivacina (3) e affinina (4), affinisina (5) e Nb-metilaffinisina (6), foram isolados do extrato metanólico das cascas das raízes de Tabernaemontana hystrix. Os triterpenos conhecidos 3-O-acetil-alfa-amirina, 3-O-acetil-beta-amirina, 3-O-acetil-lupeol foram também identificados. As estruturas dos compostos foram elucidadas com base na análise de dados espectroscópicos.

ARTICLE

A new indole alkaloid isolated from Tabernaemontana hystrix steud (Apocynaceae)

Cecilia Silva Monnerat; Jucimar Jorgeane de Souza; Leda Mathias; Raimundo Braz-Filho; Ivo José C. Vieira^*

Setor de Química de Produtos Naturais, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-602 Campos dos Goytacazes - RJ, Brazil

ABSTRACT

A new alkaloid, named hystrixnine (1), and five known indole alkaloids, ibogamine (2), olivacine (3), affinine (4), affinisine (5) and N_b-methylaffinisine (6), were isolated from the root bark of Tabernaemontana hystrix. The known triterpenes a-amyrin acetate, b-amyrin acetate and lupeol acetate were also identified. The structures of the compounds were elucidated based on spectroscopic studies.

Keywords: Tabernaemontana hystrix, Apocynaceae, indole alkaloids, triterpenes

RESUMO

Um novo alcalóide, denominado histrixnina (1), e cinco alcalóides indólicos conhecidos, ibogamina (2), olivacina (3) e affinina (4), affinisina (5) e N_b-metilaffinisina (6), foram isolados do extrato metanólico das cascas das raízes de Tabernaemontana hystrix. Os triterpenos conhecidos 3-O-acetil-a-amirina, 3-O-acetil-b-amirina, 3-O-acetil-lupeol foram também identificados. As estruturas dos compostos foram elucidadas com base na análise de dados espectroscópicos.

Introduction

Indole alkaloids exhibit numerous biological activities (such as anti-tumor, anti-microbial, anti-hypertensive and central nervous system stimulant).¹ They can be found in plants of the Apocynaceae, Rubiaceae, and Loganiaceae families.^1,2

Among the Apocynaceae, the genus Tabernaemontana is especially rich in indole alkaloids. They are useful chemical markers of the genus, and also have a great value for the classification of the individual species within the genus.³ The classification of individual species only on the basis of morphological characters has been difficult, leading to numerous synonyms.⁴

The species Tabernaemontana hystrix Steud. should have the homotypic synonym Tabernaemontana echinata Vell. and Peschiera hystrix (Steud.) A. DC., and the heterotypic synonyms T. collina Gardn. in Hooker, T. fuchsiifolia (A. DC.), Peschiera fuchsiifolia (A. DC.) Miers, T. gaudichaudii A. DC., T. lundii A. DC., Peschiera lundii (A. DC.) Miers, T. gracilis Muell., T. bracteolaris Muell., Peschiera granulosa Miers and Peschiera solandri Miers.⁴ In fact, previous phytochemical studies have been published under the name Peschiera fuchsiifolia (A. DC.) Miers.^5,6

As part of our continuing interest in the phytochemical investigation of Tabernaemontana species occurring in Brazil,^7-9 we decided to study T. hystrix, a native species of the Atlantic forest in Southeastern Brazil, popularly known as "esperta".

In the present work, we report the phytochemical analysis of the crude methanolic extract of T. hystrix, which allowed to characterize the presence of six indole alkaloids (1 to 6), including the new one named hystrixnine (1), and three triterpenoids. The structures were established by spectrometric techniques, mainly EIMS and 1D and 2D NMR, including comparative analysis with literature values.

Results and Discussion

Chromatographic purification of T. hystrix root bark methanol extract yielded triterpenes common in plants, including other Tabernaemontana species.¹⁰ The triterpene acetates were obtained as a mixture of a-amyrin acetate, b-amyrin acetate and lupeol acetate. They were identified by ¹H and ¹³C NMR spectral data compared with literature values.¹¹

The known indole alkaloids, ibogamine (2),^12,13 olivacine (3),^14,15 affinine (4),^5,16,17 affinisine (5)^5,6,17 and N_b-methylaffinisine (6)⁶ were identified on the basis of ¹H and ¹³C NMR spectral data, including homonuclear ¹H-¹H-COSY and heteronuclear ¹H-¹³C 2D shift-correlated NMR experiments, which were also used to complete and unambiguous ¹H and ¹³C chemical shift assignments.¹⁸

The UV spectrum of hystrixnine (1) showed absorptions at l_max 223 and 282 nm (e 42566 and 6287, respectively) typical of an substituted indole chromophore,⁸ while the IR spectrum revealed bands at n_max 3360 (N-H), 1736 (conjugated carbonyl ketone group stretching), 2930-2830 (C-H stretching) and 1616, 1591 and 743 cm^-1 (C-H bending of benzene ring).⁸ The EIMS showed a molecular peak at m/z 338 daltons ([M]^.+) which together with ¹H and ¹³C NMR spectral data (Table 1) allowed to deduce the molecular formula C₂₁H₂₆N₂O₂ (ten degrees of unsaturation) compatible with corynanthean skeleton.⁸ The principal peaks observed in the EIMS spectrum are in agreement with proposed fragmentation mechanisms summarized in Scheme 1.

Carbon-13 NMR experiments ({¹H} and APT) revealed the presence of three methyl groups, four methylenes (sp³), eight methines (three sp³ and five sp²) and six (sp²) quaternary carbon atoms. The ¹H-¹H-COSY, HMQC and HMBC experiments established geminal and vicinal hydrogen interactions as well as direct (¹J_CH) and two and three bond correlations between carbon and hydrogen atoms in the structure (Table 1). These data revealed that 1 is closely related to affinine (4), differing by the presence of methoxyl group linkage at C-17. The presence of the indole nucleus was clearly indicated by the ¹H and ¹³C aromatic signals (Table 1). Typically the ¹H NMR revealed two singlet signals at d_H 3.47 (MeO-17) and 2.57 (MeN-4) and double doublet signal at d_H 1.70 (J= 7.0 and 2.2 Hz, 3H-18 linkage at sp² CH-19) corresponding to methyl groups. Through analysis of the HMBC spectrum these signals were assigned by corresponding cross-peaks, due to heteronuclear spin-spin coupling via three (³J_CH) bonds, two methyl groups linked to the allylic Me-18 and N-4 (aliphatic N_b), respectively: i) Me-18 (d_H 1.70) with C-20 (d_C 134.71); ii) and MeN-4 (d_H 2.57) with both CH-5 (d_C 56.93) and CH₂-21 (d_C 52.04). The ketone group localized at position C-3 was confirmed by correlations with H-14b [d_H 3.33 (²J_CH)] and H-15[(d_H 3.07 (³J_CH)]. The presence of methoxyl group was confirmed by ¹H NMR and ¹³C NMR spectra by presence of the signals at d_H 3.47 (s) and d_C 50.74 (Table 1). The complete analysis of this HMBC spectrum in combination with additional NMR spectral data also allowed the identification of a skeleton as that of the indole alkaloid affinine (4)^5,16,17 and the total ¹H and ¹³C chemical shift assignments, as summarized in Table 1. Thus, the new alkaloid corynanthean skeleton isolated from Tabernaemontana hystrix was characterized as 1, named hystrixnine.

In accordance with the revision published by Leeuwenberg,⁴ the alkaloid series isolated in this study from T. hystrix are closely related to those previously reported from Peschiera fuchsiifolia: decarbomethoxyvoamine, demethylvoacamine, voacamidine, perivine, 16-epiaffinine, voacangine hydroxyindolenine, fuchsiaefoline, 12-methoxy-N_b-methylvoachalotine and 12-methoxy-N_b-methylvoachalotine ethyl ester (reported by Braga and co-workers).^5,6 The similarity of the alkaloids reported in this work in comparison with those from two other Brazilian Tabernaemontana species is remarkable. From T. solanifolia were reported the alkaloids: isovoacangine, isovoacristine, coronaridine, voacangine, voacangine hydroxyindolenine, heyneanine, voacamine, voachalotine and 12-methoxy-Nb-methylvoachalotine [reported under the name Peschiera campestris (Rizz.) Rizz. by Gower et al.]¹⁹ and P. laeta were described the alkaloids: coronaridine, voacangine, isovoacangine, 19-(S)-heyneanine, isovoacristine, 3-oxoisovoacangine, ibogaine, iboxygaine, tabersonine, apparicine, vobasine, N_b-methylvoachalotine, voacamine, conodurine and tabernamine [reported under the name T. laeta (Mart.) by Medeiros and co-workers].^7-9 This similarity might point to a close taxonomic relationship of these recognized species.

Experimental

General

¹H NMR and ¹³C NMR: At Jeol Eclipse spectrometer operating at 400 MHz and 100 MHz, respectively, in CDCl₃, using the residual solvent signals as internal standard (Table 1).

Plant materials

The root bark of Tabernaemontana hystrix Steud. was collected in March 2002 at Varre e Sai, Rio de Janeiro State, Brazil, and identified by Dr. A. J. M. Leeuwenberg of the Agricultural University of Wageningen, The Netherlands. A voucher specimen (WAG) is deposited at the herbarium of the Agricultural University of Wageningen, Netherlands.

Extraction and isolation

Dried and powdered root bark (0.92 kg) from T. hystrix Steud. was extracted at room temperature using methanol, furnishing after solvent evaporation, crude methanol extracts (40.0 g).

23.0 g of the methanol extract was chromatographed on a Si gel column and eluted with a gradient of MeOH in CH₂Cl₂, yielding 11 fractions. The fractions 1-3 (460 mg) was recrystallizated from hexane to furnish a mixture of the three triterpenes (180 mg) a-amyrin acetate, b-amyrin acetate and lupeol acetate; fraction 5 (940 mg) furnished 2 (58 mg); fraction 8 (1.58 g) was rechromatographed on a Si gel column using a gradient of MeOH in CH₂Cl₂ affording 5 (73 mg); fraction 9 (1.36 g) was rechromatographed in the same way, yielding the alkaloids 3 (73 mg), 4 (26 mg) and 6 (11 mg). 2.6 g of fraction 10 was rechromatographed on a Si gel column using a gradient of MeOH in CH₂Cl₂ furnishing 06 fractions, of which, fraction 4 (54 mg) furnished the alkaloid 1 (7.9 mg) after rechromatography with a mixture of MeOH in CH₂Cl₂.

The four alkaloids 2-6, as well as three triterpenes were identified by the analysis of ¹H and ¹³C NMR and comparison with literature values.^11-17

hystrixnine (1). Amorphous solid; [a]²⁵_D - 100º (MeOH, c 0.66); IR (KBr) n_max/cm^-1 1736 (C=O), 1616, 1591, 743 (aromatic ring); UV l_max/nm (CH₃OH) 223 (e 42566), 282 (e 6287). EIMS: Scheme 1; ¹H NMR and ¹³C NMR: Table 1.

Acknowledgements

The authors are grateful to Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for grants and to Conselho Nacional de Desenvolvimento Científico (CNPq-Brazil) for a research fellowship and grants.

Received: June 30, 2005

Published on the web: October 6, 2005

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Publication Dates

• Publication in this collection
  20 Jan 2006
• Date of issue
  Nov 2005

History

• Received
  30 June 2005