Trypanocidal Activity of Oxoaporphine and Pyrimidine-β-Carboline Alkaloids from the Branches of Annona foetida Mart. (Annonaceae)

Phytochemical investigation of the branches of Annona foetida Mart. led to isolation from the CH2Cl2 extract of four alkaloids: Atherospermidine (1), described for the first time in this species, liriodenine (2), O-methylmoschatoline (3), and annomontine (4). Their chemical structures were established on the basis of spectroscopic data from IR, MS, NMR (1D and 2D), and comparison with the literature. Compounds 2–4 showed potent trypanocidal effect when evaluated against epimastigote and trypomastigote forms of Trypanosoma cruzi.


Introduction
Annona L. belongs to the family Annonaceae and comprises approximately 175 species of trees and shrubs found predominantly in lowland tropical regions [1]. Economically, this genus is the most important of the Annonaceae family due to its edible fruits and medicinal properties [2]. Previous chemical and pharmacological investigations on some species of this genus have indicated the presence of important bioactive compounds, exhibiting various pharmacological activities including cytotoxicity against various tumor cell lines [3][4][5], antimicrobial [6,7], antioxidant [6], antiplatelet [8,9], and antiparasitic properties, in particular against Leishmania sp. [7,[10][11][12] and Trypanosoma cruzi [10,12,13]. These activities generally are attributed to the presence of alkaloids, acetogenins, and terpenes. In Brazil this genus contain close to 60 species, with the largest part occurring in forests and few representatives in open areas.
Annona foetida Mart. is a 3-15 m tall tree found in the Brazilian and Peruvian Amazon region. It is popularly known as "envira-ata", "envireira", "araticum caatinga" and "graviola da mata" [2,14]. In folk medicine the leaves and bark decoction are used for treatment of rheumatism, intermittent fevers and ulcers [2]. Previous studies on this species describe the isolation of oxoaporphine and pyrimidineβ-carboline alkaloids [11], and essential oils with antileishmanial and antimicrobial activities [7]. As a result of our continuing study on A. foetida in a search for antiparasitic natural products, mainly against T. cruzi, we report herein the phytochemical and biological study of the CH 2 Cl 2 extract from the branches of this species.

Results and Discussion
Phytochemical investigation of CH 2 Cl 2 extract from the branches of Annona foetida led to the isolation of four alkaloids (Figure 1), namely three oxoaporphine atherospermidine (1), liriodenine (2) and O-methylmoschatoline (3) and one pyrimidine-β-carboline one, annomontine (4).   The structural elucidation of these alkaloids was carried out based on their spectroscopic data, mainly 1D and 2D NMR, and also comparison with the literature data [11,13,[15][16][17]. Atherospermidine (1) is described for the first time in this species, while compounds 2-4 have been described in the bark. Atherospermidine (1), liriodenine (2), and O-methylmoschatoline (3) are three oxoaporphine alkaloids widely found in almost all the genera of the family Annonaceae. However, the pyrimidine-β-carboline alkaloid annomontine (4) has been described only in the genus Annona [11,18,19]. Natural products or derivatives play an important role in the development of all types of drugs and some natural compounds or extracts have shown trypanocidal activity [20]. The compounds liriodenine (2), O-methylmoschatoline (3), and annomontine (4) were investigated for their biological activity against epimastigote and trypomastigote forms of T. cruzi ( Table 1). The comparison of the 50% inhibitory concentration values (IC 50 in µg/mL) revealed that all three evaluated compounds showed some activity against epimastigote forms, with O-methylmoschatoline (3) being the most active isolated compound, with an IC 50 value of 92.0 ± 18.4 µg/mL. The presence of 3 in the culture of epimastigotes caused progressive parasite injury, compared to untreated cells, and a dose-dependent effect was also observed. After 96 h of incubation with the concentration of 500 µg/mL, parasite growth was completely arrested. Table 1. Trypanocidal activity of the oxoaporphine and pyrimidine--carboline alkaloids (2)(3)(4).

General
UV spectra were obtained in CH 3 OH on a UV-Vis Agilent HP 8453 spectrophotometer. IR spectra were acquired on a Bomem MB-100 spectrophotometer. 1D and 2D NMR experiments were acquired in CDCl 3 , CDCl 3 + CD 3 OD or CD 3 OD at 293 K on a Bruker AVANCE 400 NMR spectrometer operating at 9.4 Tesla, operating at 400 and 100 MHz for 1 H and 13 C at, respectively. The spectrometer was equipped with a 5-mm multinuclear direct detection probe with z-gradient. One-bond and longrange 1 H-13 C correlation (HSQC and HMBC) experiments were optimized for an average coupling constant 1 J (C,H) and LR J (C,H) of 140 and 8 Hz, respectively. All 1 H-and 13 C-NMR chemical shifts () are given in ppm related to the TMS signal at 0.00 ppm as internal reference, and the coupling constants (J) in Hz. Low-resolution ESI-MS data were taken in the positive ion mode, on a Micromass Quattro LC mass spectrometer. Silica gel 60 (70-230 mesh) was used for column chromatography, while silica gel 60 F 254 were used for analytical (0.25 mm), and preparative (1.00 mm) TLC. Compounds were visualized by exposure under UV 254/366 light, spraying p-anisaldehyde reagent followed by heating on a hot plate, as well as spraying with Dragendorff's reagent.

In Vitro Trypanocidal Assay
Parasites: Epimastigote forms of Trypanosoma cruzi strain Y were grown in Liver Infusion Tryptose (LIT) supplemented with 10% fetal-calf serum (FCS, Gibco) at 28 °C for 96 h.
Antiproliferative activity on epimastigote forms: Epimastigote forms of T. cruzi in the logarithmic phase were used for this assay. The compounds liriodenine (2), O-methylmoschatoline (3) and annomontine (4) were dissolved in DMSO and LIT medium to obtain concentrations of 1, 5, 10, 50, 100, 500 and 1,000 µg/mL, in different wells. The final concentration of DMSO did not exceed 1%.
A cell density of 1 × 10 6 epimastigotes/mL was cultured in a 24-well microplate to obtain a final volume of 1 mL. The cells were incubated at 28 °C and their growth was determined by counting the parasites with a hemocytometer chamber after 96 h. Benznidazole was used as the reference drug. The IC 50 value (50% inhibitory concentration) was determined using linear regression analysis from this inhibitory percentage. These tests were performed in triplicate on separate occasions.
Activity against trypomastigote forms: The tissue-culture-derived trypomastigotes were resuspended in Dulbecco's modified Eagle medium supplemented with FCS containing 10% mouse blood in a concentration of 10 7 parasites/mL. In a 96-well microplate, 100 µL of this suspension was added to the same volume of the compounds diluted in DMSO and DMEM at twice the desired final concentration (1, 5, 10, 50, 100, 500 and 1000 µg/mL), and incubated for 24 h at 37 °C. We used the Pizzi-Brener method to appreciate the mobility, thus the viability of this form of parasite. For this an aliquot of 5 µL of each sample were placed on slides plus coverslips and immediately counted in an optical microscopy [23], subsequently the EC 50 (concentration which lysed 50% of the parasites) was calculated. Crystal violet was used as the reference drug. from the branches of Annona foetida, compound 1 being described for the first time in this species. These finds contribute to the chemotaxonomy of the family Annonaceae, especially for the genus Annona. Trypanocidal activity assays revealed that all alkaloids tested (compounds 2-4) shown strong trypanocidal effects against the clinical relevant trypomastigote forms of T. cruzi, even better than positive control. The oxoaporphine alkaloid O-methylmoschatoline (3) was the most active for both epimastigote and trypomastigote forms. This results support further evaluations over analogs, as well as encourages in vitro and in vivo investigations of these alkaloid kinds.