A New Natural Quaternary Indole Alkaloid Isolated from Tabernaemontana laeta Mart . ( Apocynaceae )

Um novo alcalóide quaternário natural, N b -metilvoachalotina (1), três alcalóides indólicos diméricos, conodurina (2), voacamina (3) e tabernamina (4), e os três alcalóides monoméricos 19S-heyneanina (5), coronaridina (6) e voacangina (7) foram isolados da casca das raízes de Tabernaemontana laeta. Os triterpenos conhecidos 3-O-acetil-amirina, 3-O-acetil-amirina, 3-O-acetil-lupeol e 3-O-acetiltaraxasterol e os fitoesteróides -sitosterol e seu derivado 3-O-Dglicopiranosídeo 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 laeta Mart.has the homotypic synonym Peschiera laeta (Mart.)Miers, and the heterotypic synonyms T. laeta var.pubiflora Muell.Arg., T. laeta var.minor Muell.Arg., T. laeta var.densa Muell.Arg., T. spixiana Mart.ex Muell.Arg. and T. breviflora Muell.Arg. 4 .Some previous phytochemical studies have been published under the name Peschiera laeta Mart. 5,6.
As part of our continuing interest in the phytochemical investigation of Tabernaemontana species occurring in Brazil, we decided to study T. laeta, a native species of the Atlantic forest in Southeastern Brazil.Popularly known as "esperta" or "guarana", this species is generally considered poisonous.In a recent paper, we reported the complete 1 H and 13 C chemical shift assignments of the dimeric indole alkaloids conodurine (2) and voacamine (3); both isolated from its methanolic extracts 7 .
In the present study, we report the phytochemical analysis of the crude extracts of T. laeta, including the isolation of the new alkaloid N b -methylvoachalotine (1).

Results and Discussion
Chromatographic purification of T. laeta root bark hexane and methanol extracts yielded triterpenes and phytosterols common in plants, including other Tabernaemontana species 8 .The triterpene acetates were obtained as a mixture of a-amyrin acetate, b-amyrin acetate, lupeol acetate and taraxasterol acetate.They were identified by comparison of the 1 H and 13 C NMR spectral data with literature values 9 .The phytosterol b-sitosterol, isolated from the hexane extract, and its 3-O-b-D-glucopyranosyl derivative, isolated from the methanol extract, were also identified by comparison with published 1 H and 13 C NMR spectral data, including the data of the peracetyl derivative of 3-O-b-D-glucopyranosyl-b-sitosterol 10 .
The 19S configuration shown in 5 was clearly established by comparison of the NMR chemical shifts obtained with those reported for heyneanine (5) and its epimer, (19R)-epiheyneanine (8).Due to hydrogen bonding between the hydroxyl group at C-19 and the nearby nitrogen the 1-hydroxyethyl moiety has a rigid conformation (see partial structures 5a and 8a), leading to considerable chemical shift differences.In the 1  The new indole alkaloid 1 was isolated from the methanol extract.The ESMS showed a molecular ion at m/z 381 daltons ([M] .+), in agreement with the molecular formula C 23 H 29 N 2 O 3 and with 1 H and 13 C NMR spectral data.This alkaloid was nearly insoluble in chloroform and its NMR spectra were therefore obtained in DMSO-d 6 .Carbon-13 NMR experiments (HBBD and DEPT) revealed the presence of four methyl groups, four methylenes (sp 3 ), eight methines (three sp 3 and five sp 2 ) and seven quaternary carbon atoms (one sp 3 and six sp 2 ).The 1 H-1 H-COSY, HMQC and 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  7 .Thus, the new quaternary indole alkaloid isolated from Tabernaemontana laeta was characterized as N b -methylvoachalotine (1), a new natural product.
Although it has been synthesized from voachalotine in a previous study 18 , only the 13 C NMR data of the iodide salt were reported.
In accordance with the revision published by Leeuwenberg 4 , the alkaloid series isolated in this study from T. laeta are closely related to those previously reported from Peschiera laeta: geissoschizol, affinine, akuammidine, normacusine B, vobasine, conodurine and voacamine 5,6 .The similarity of the alkaloids found in this work in comparison with those from two other Brazilian Tabernaemontana species is remarkable.From T. solanifolia were reported the following 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, Pereira and Marsaioli 19 ], while from T. hystrix were reported the following alkaloids: 12-methoxy-N b -methylvoachalotine, 12-methoxy-N b -methylvoachalotine ethyl ester and fuchsiaefoline (reported under the name Peschiera fuchsiaefolia by Braga and Reis 18 ).This similarity might point to a close taxonomic relationship of these recognized species.

Experimental
General 1 H NMR and 13 C NMR spectra were obtained with Bruker DRX spectrometers operating at 400 and 600 MHz and 100 and 150 MHz, respectively, in CDCl 3 or DMSOd 6 , using the residual solvent signals as internal standard (Table 1).

Plant material
The root bark of Tabernaemontana laeta Mart.was collected in July 1997 at Bom Jesus do Itabapoana, 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 (1.06 kg) from T. laeta Mart.was extracted at room temperature using hexane and methanol, furnishing, after solvent evaporation, crude hexane (33.4 g) and methanol (120.0 g) extracts, respectively.
The hexane extract was purified by silica gel chromatography, with a gradient of EtOAc in hexane to give four fractions.The second fraction supplied large amounts (28.0 g) of a product, which by recrystallization in hexane, furnished a mixture of the four triterpenes, aamyrin acetate, b-amyrin acetate, lupeol acetate and taraxasterol acetate.The third fraction yielded, after recrystallization, b-sitosterol (220.0 mg).The fourth fraction yielded, after column chromatography with the same system, the alkaloid voacangine 13  (7, 15.0 mg).
The methanol extract was redissolved in MeOH/H 2 O (3:1) and extracted with CHCl 3 .The CHCl 3 layer was evaporated and further partitioned between MeOH and hexane (1:1).After another extraction with hexane, the hydromethanolic fraction was extracted with EtOAc.The methanol phase yielded 55.0 g upon evaporation, the hexane phase 22.4 g and the EtOAc phase 2.0 g.

Table 1 .
13 (400 MHz) and13C (100 MHz) NMR for N b -methylvoachalotine(1), including results obtained by heteronuclear 2D shift-correlated HMQC ( 1 J CH ) and HMBC ( n J CH , n=2 and 3), in DMSO-d 6 as solvent and residual DMSO-d 6 used as internal reference (d H 2.50 and d C 39.50).Chemical shifts (d, ppm) and coupling constants (J, Hz, in parenthesis).*tht 1 is closely related to voachalotine (vide infra), differing by the presence of an additional methyl group.The presence of the indole nucleus was clearly indicated by the 1 H and13C aromatic signals (Table1).Typically the *Number of hydrogens bound to carbon atoms deduced by comparative analysis of HBBD-and DEPT-13 C NMR spectra.Chemical shifts and coupling constants (J) obtained of 1D 1 H NMR spectrum.Superimposed 1 H signals are described without multiplicity and chemical shifts deduced by HMQC, HMBC and 1 H-1 H-COSY spectra.revealed 1 H and 13 C chemical shift assignments, as summarized in Table 1.The stereochemistry of the chiral carbon atom C-16 shown in 1 was clearly indicated by the chemical shift of the signal at d H 3.67 corresponding to the hydrogen of the MeO-22 (carbomethoxy group).In its epimer this signal should appear at about d H 2.50, as observed in 2 and 3 by singlet signals of the MeO-22' at d H 2.51 and 2.46, respectively