A Pyrimidine--carboline and Other Alkaloids from Annona foetida with Antileishmanial Activity Emmanoel V. Costa, † Maria Lu ´cia B. Pinheiro,* ,† Clahildek M. Xavier, † Jefferson R. A. Silva, † Ana Cla ´udia F. Amaral, ‡ Afonso D. L. Souza, † Andersson Barison, ⊥ Francinete R. Campos, ⊥ Antonio G. Ferreira, ⊥ Ge ´rzia M. C. Machado, § and Leonor L. P. Leon § Departamento de Quı ´mica, UniVersidade Federal do Amazonas, AVenida Gen. Rodrigo Ota ´ Vio Jorda ˜ o Ramos, 3000, Coroado, CEP 69077-000, Manaus, Amazonas, Brazil, Instituto de Tecnologia em Fa ´ rmacos, Departamento de Produtos Naturais, FIOCRUZ, Rua Sizenado Nabuco, 100, Manguinhos, 21041-250, Rio de Janeiro, Rio de Janeiro, Brazil, Departamento de Imunologia, IOC, FIOCRUZ, AVenida Brasil, 4365, Manguinhos, 21045-900, Rio de Janeiro, Rio de Janeiro, Brazil, and Departamento de Quı ´mica, UniVersidade Federal de Sa ˜ o Carlos, 13565-905, Cx. Postal 676, Sa ˜ o Carlos, Sa ˜ o Paulo, Brazil ReceiVed October 22, 2005 Bioassay-guided fractionation of the bark extract of Annona foetida afforded a new antileishmanial pyrimidine--carboline alkaloid, N-hydroxyannomontine (1), together with the previously reported annomontine (2), O-methylmoschatoline (3), and liriodenine (4). The structure of compound 1 was established on the basis of extensive 1D and 2D NMR and MS analyses. This is the third reported pyrimidine--carboline-type alkaloid and is particularly important for Annona genus chemotaxonomy. In addition, all compounds exhibit in vitro antileishmanial activity against promastigote forms of Leishmania braziliensis. Compounds 2 and 4 showed better activity than compounds 1 and 3 against L. braziliensis. Compound 2 was not active against L. guyanensis. Plants of the Annonaceae family are known as a rich source of aporphinic alkaloids. Previous chemical and pharmacological investigations on species in this family have indicated the presence of important bioactive compounds, exhibiting various pharmaco- logical activities including antiparasitic, in particular against Leishmania sp., 1-5 Plasmodium falciparum, 6,7 and Trypanosoma cruzi. 1,5 Therefore, in a search for novel antiparasitic natural products we have studied the bark of Annona foetida Martius. This annonaceous plant, known commonly as “envira-ata” and “graviola do mato”, is a tropical native tree found in the Brazilian and Peruvian Amazon. 8 Its seeds are traditionally used in Brazil as an insecticide and antiparasitic. The present paper reports the isolation and characterization of a new pyrimidine--carboline alkaloid, named N-hydroxyannomontine (1), together with previously re- ported annomontine (2) 9 and the oxoaporphinic alkaloids O- methylmoschatoline (3) 10 and liriodenine (4). 10 The antiparasitic activity was evidenced for the crude extract and investigated for each purified compound on Leishmania braziliensis and L. guy- anensis, the main causes of leishmaniasis in the Brazilian state of the Amazon. Having discovered that the CH 2 Cl 2 and MeOH crude extracts possessed in vitro activity against L. braziliensis (IC 50 23.0 and 40.4 µg/mL, respectively) and L. guyanensis (IC 50 2.7 and 23.6 µg/mL, respectively), as shown by screening tests (Table 1), bioassay-guided fractionation of the extracts was undertaken as described in the Experimental Section, which led to the isolation and identification of four alkaloids, namely, two pyrimidine-- carboline alkaloids (1 and 2) and two oxoaporphine alkaloids (3 and 4). The structures of these compounds were elucidated by spectroscopic methods, including 1D and 2D NMR and MS analyses. Compound 1 was obtained as red needles and has the molecular formula C 15 H 11 N 5 O, as deduced from its HREIMS (observed m/z 277.09469) and NMR data. The IR spectrum showed broad absorption bands due to O-H/N-H groups (3420 and 3172 cm -1 ) and typical absorption for aromatic rings (1600-1450 cm -1 ). The UV spectrum revealed a conjugated aromatic chromophore with maxima at 246, 312, 398, and 428 nm. The ESIMS of 1 showed a protonated molecule at m/z 278.5 [M + H] + , 16 Da higher than the pyrimidine--carboline alkaloid annomontine (2). This suggested the presence of a hydroxyl group in the structure of 1, which was evidenced in the IR spectrum and NMR data. Additionally, the ESIMS/MS (70 eV) experiments of 1 gave informative fragment * To whom correspondence should be addressed. Tel/Fax: +55-92- 32322442. E-mail: lbelem@ufam.edu.br. † Universidade Federal do Amazonas. ‡ Departamento de Produtos Naturais, Fundac ¸ a ˜o Oswaldo Cruz. § Departamento de Imunologia, Fundac ¸ a ˜o Oswaldo Cruz. ⊥ Universidade Federal de Sa ˜o Carlos. Table 1. In Vitro Activity of Annona foetida Extracts and Compounds against Leishmania Species (promastigote forms) a IC50 (µg/mL) extract/fraction L. braziliensis L. guyanensis hexane >160 42.7 ( 5.4 CH 2Cl2 23.0 ( 0.6 2.7 ( 0.4 CH2Cl2 alkaloidal fraction 18.3 ( 2.5 10.3 ( 0.9 MeOH 40.4 ( 3.2 23.6 ( 3.1 MeOH alkaloidal fraction 24.3 ( 1.9 9.1 ( 0.8 IC50 (µM) compound L. braziliensis L. guyanensis N-hydroxyannomontine (1) 252.7 ( 2.2 437.5 ( 2.5 annomontine (2) 34.8 ( 1.5 >613.0 O-methylmoschatoline (3) 320.8 ( 3.1 103.7 ( 3.4 liriodenine (4) 58.5 ( 1.8 21.5 ( 0.4 pentamidine b 2.9 ( 0,3 0.9 ( 0.3 a The IC50 values are expressed as mean ( SEM of three determina- tions. b Standard antileishmanial agent. 292 J. Nat. Prod. 2006, 69, 292-294 10.1021/np050422s CCC: $33.50 © 2006 American Chemical Society and American Society of Pharmacognosy Published on Web 02/09/2006 Downloaded via FIOCRUZFUNDACAO OSWALDO CRUZ on December 26, 2019 at 17:15:01 (UTC). 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