A New Indole Alkaloid Isolated from Tabernaemontana hystrix Steud (Apocynaceae)

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 3O-acetil-α-amirina, 3-O-acetil-β-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 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 α-amyrin acetate, β-amyrin acetate and lupeol acetate. They were identified by 1 H and 13 C 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 1 H and 13 C NMR spectral data, including homonuclear 1 H-1 H-COSY and heteronuclear 1 H-13 C 2D shift-correlated NMR experiments, which were also used to complete and unambiguous 1 H and 13 C chemical shift assignments. 18 The UV spectrum of hystrixnine (1) showed absorptions at λ max 223 and 282 nm (ε 42566 and 6287, respectively) typical of an substituted indole chromophore, 8 while the IR spectrum revealed bands at ν 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 1 H and 13 C NMR spectral data (Table 1) allowed to deduce the molecular formula C 21 H 26 N 2 O 2 (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 ({ 1 H} and APT) revealed the presence of three methyl groups, four methylenes (sp 3 ), eight methines (three sp 3 and five sp 2 ) and six (sp 2 ) quaternary carbon atoms. The 1 H-1 H-COSY, HMQC and A New Indole Alkaloid Isolated from Tabernaemontana hystrix Steud Vol. 16, No. 6B, 2005 HMBC experiments established geminal and vicinal hydrogen interactions as well as direct ( 1 J 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 1 H and 13 C aromatic signals (  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 1 H and 13 C 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: decarbomethoxy-  voamine, 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

Experimental
General 1 H NMR and 13 C NMR: At Jeol Eclipse spectrometer operating at 400 MHz and 100 MHz, respectively, in CDCl 3 , using the residual solvent signals as internal standard (Table 1).

Plant materials
The root bark of Tabernaemontana

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 CH 2 Cl 2 , yielding 11 fractions. The fractions 1-3 (460 mg) was recrystallizated from hexane to furnish a mixture of the three triterpenes (180 mg) α-amyrin acetate, β-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 CH 2 Cl 2 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 CH 2 Cl 2 furnishing 06 fractions, of which, fraction 4 (54 mg) furnished the alkaloid 1 (7.9 mg) after rechromatography with a mixture of MeOH in CH 2 Cl 2 .