Antiprotozoal Alkaloids from Psychotria prunifolia (Kunth) Steyerm

A continuidade do estudo fitoquímico de P. prunifolia com a análise dos extratos etanólicos obtidos a partir de suas raízes e galhos levou ao isolamento de cinco alcaloides indol-b-carbolínicos dos quais, dois derivados, o 10-hidróxi-iso-deppeaninol e o N-óxido-10-hidróxi-antirhina são descritos pela primeira vez. As estruturas foram determinadas por análise de técnicas espectroscópicas de IV, EMAR e RMN (H e C, 1D e 2D). A avaliação da atividade frente à Leishmania amazonensis e Trypanosoma cruzi, mostrou que os extratos brutos e os alcaloides 14-oxoprunifoleína e estrictosamida inibiram as formas promastigotas de L. amazonensis, com valores de CI50 de 16,0 e 40,7 mg per mL, respectivamente.


Introduction
The genus Psychotria, one of the largest genera of the Rubiaceae, has a long history of indigenous use as a component of the hallucinogenic beverage ayahuasca and also in traditional medicines used to treat microbial infections, inflammatory disease and complications of pregnancy.Pharmacological studies of Psychotria species, such as P. umbelata Tonn, P. leiocarpa Cham.][3][4][5] P. prunifolia is an understory shrub ranging from Venezuela, throughout the Amazon basin, to Bolivia and southern Brazil, with its southern limit in the state of São Paulo.7][8] In our laboratory, phytochemical study of the leaves of P. prunifolia collected in Brazilian Cerrado have led to the isolation of b-carboline alkaloids and triterpenes. 9umerous reports in the current literature provide evidence that alkaloids possessing an indole moiety could display important antiprotozoal activities.For example, alkaloids like harmane have exerted antiproliferative effects toward parasites of the genus Trypanosoma. 10,11he bis-indole alkaloids ramiflorines A and B isolated from Aspidosperma ramiflorum exhibited remarkable activity in in vitro assays against promastigote forms of L. amazonensis. 12In addition, indole alkaloids isolated from the bark of Corynanthe pachyceras (Rubiaceae) and Kopsia griffithii (Apocynaceae) showed activity against promastigotes of L. major and L. donavani. 13,14n this study, further phytochemical work was performed on P. prunifolia with particular attention to their alkaloid constituents, resulting in the isolation of two new beta carboline derivatives (1 and 3), besides the three known compounds 2, 4, and 5.Here we report the structural determination of the new alkaloids and the antiprotozoal activity of the crude extracts of P. prunifolia and the major alkaloids 4 and 5.

Structural elucidation of new alkaloids
Alkaloid 1 [a] 25 = -21.5º(MeOH, c = 0.085) was obtained as a yellowish oil and was positive with Dragendorff`s reagent.Analysis of HRMS data indicated that compound J HMBC correlation observed between H-14/C-3 and 3 J observed for H-14/C-16 and H-14/C-20 permitted us to suggest the connection between the aliphatic unit and the indolic b-carboline unit (Figure 1).Further HMBC correlations are described in Table 1.All these data were consistent with a new alkaloid similar to 10-hydroxy derivative of deppeaninol, which was assigned as alkaloid 1 identified as 10-hydroxy-iso-deppeaninol.The relative configuration proposed is that observed of corynantheineheteroyohimbine alkaloids.Deppeaninol was isolated from Deppea blumenaviensis (Rubiaceae) and described by Kan-Fan et al. 15 Alkaloid 2 was obtained as a brownish oil and was positive to Dragendorff´s reagent.Its molecular formula was determined by HRMS, which exhibited a molecular ion [M+H] + observed at m/z 313.1920 (calculated m/z 313.1916,C 19 H 25 N 2 O 2 ) consistent with a molecular formula of C 19 H 24 N 2 O 2 (m/z 312.1838).The 1 H (1D and 2D COSY), 13 C ({ 1 H}, DEPT 90º and DEPT 135º), HMQC and HMBC NMR spectra data showed the presence of six sp 3 methylene units (one of which was oxygenated), two olefinic carbons (one of which was a methylene unit), three aromatic and three aliphatic methines, and five sp 2 quaternary carbons.The resonances and the 1 H NMR J values at d H 6.80 (d, J 2.4 Hz), 6.68 (dd, J 8.7 and 2.4 Hz) and 7.17 (d, J 8.7 Hz) were attributed to a trisubstituted aromatic system with a 10 or 11-substitution pattern.
In addition to these assignments, the HMBC correlations from H-5a (d H 3.57, dd, J 12.6 and 6.0 Hz) to C-7 (d C 106.0) and from H-6a (d H 2.82, dd, J 16. Analysis of COSY data indicated proton spin systems corresponding to H-16/H-17, H-3/H-14/H-15 and H-18/H-19/H-20/H-21, suggesting a cyclic terpene scaffold.HMBC correlations from H-18 to C-20 and H-21 to C-20/C-15 further revealed the connectivities in the terpene unit.The 3 J HMBC correlations among H-6 to C-2, H-5 to C-17 and H-14 to C-2 led to the direct connection of the indole and terpene moieties.All of these assignments, combined with the literature data, permitted us to identify alkaloid 2 as 10-hydroxy-antirhine, which was previously isolated from Ochrosia alyxiodis. 16-20, establishing a connection between the terpene and indole units.The analysis of all the spectral data suggested that 3 possessed the same skeleton of 2, but with differences in the chemical shifts at the carbons C-3, C-5 and C-17 of Dd +14.6, +16.6 and +11.1, respectively.This deshielding effect observed in the 13 C could be attributed to the influence of an N-oxide at the N-4 position and permitted us to assign alkaloid 3 as a 10-hydroxyantirhine N-oxide derivative.The alkaloids 4 and 5 were isolated previously 9 and identified as 14-oxoprunifoleine and strictosamide, respectively.

Antiprotozoal assay
In this study, the crude extracts of roots and branches and the major alkaloids 4 and 5 were evaluated for in vitro antiprotozoal activity against promastigotes of Leishmania amazonensis and epimastigotes of Trypanosoma cruzi strains.Table 3 summarises the antiprotozoal activity data for the ethanolic crude extracts and the known alkaloids (4 and 5).In both cases, L. amazonensis was the most sensitive protozoan.The ethanolic root extract was the most active among the extracts assayed.For comparison, the two

Plant material
Fresh material of P. prunifolia was collected in September 2007 in the municipality of Goiânia and identified by Piero Delprete of the Federal University of Goiás, at Bosque A. Saint-Hilaire.The plants were found in understory vegetation of seasonal semi-deciduous forest, at 16°36'12"S, 49°15'41"W and 850 m altitude.The voucher specimen, Delprete 10323, was deposited at the Herbarium (UFG) of the Federal University of Goiás, Goiânia. 9

Extraction and isolation
The air-dried and powdered branches (149 g) were successively extracted with EtOH.The resulting extract was filtered and concentrated under reduced pressure to yield 17 g.An amount of 12 g of the resulting ethanolic extract was added to 10% aq.HOAc (100 mL).The resulting suspension was incubated at 5 °C overnight.The suspension was then filtered, and the acidic aqueous phase was extracted with CHCl 3 (3 × 150 mL).The combined organic layers were treated with Na 2 SO 4 and filtered, affording a CHCl 3 acidic fraction (0.8 g -fraction A).
The aqueous layer was adjusted to pH 8-9 with a saturated aq.NaHCO 3 solution, and then again extracted with CHCl 3 (3 × 150 mL).The combined organic layers were treated with Na 2 SO 4 and filtered, affording CHCl 3 basic fractions (0.2 g -fraction B).This process was repeated using EtOAc as solvent to afford an EtOAc basic fraction (1.35 g -fraction C).
The crude extract of roots (9 g) was obtained by extraction with ethanol from 150 g of powdered roots.Then 5 g of this ethanolic extract was submitted to the same acid-base treatment described above and yielded CHCl 3 acid fraction (0.06 g -fraction D), CHCl 3 basic (0.04g -fraction E) and AcOEt basic fraction (0.1g -fraction F).Preparative TLC from fraction F (61 mg) using CH 2 Cl 2 -MeOH (20-1) as eluent system furnished 5 (8.5 mg).

General procedures
IR spectra were recorded with a FTIR Bomem MB100 using KBr pellets.Optical rotations were obtained with a Bellingham+Stanley Ltd ADP 440.NMR spectra were recorded with a Varian Mercury spectrometer operating at 300.1 MHz for 1 H and at 75.5 MHz for 13 C. CD 3 OD was used as the solvent, with Me 4 Si (TMS) used as the internal standard.HRMS was performed with a Synapt HDMS spectrometer in positive (or negative) ionisation modes of electrospray ionisation (ESI) (Waters Corporation).TLC was conducted using precoated Kiesegel 60 F 254 plates (Merck and M. Nagel).The spray developing reagents used for TLC were 50% H 2 SO 4 in CH 3 OH and Dragendorff´s reagent.

Antiprotozoal assay 17
The effects of the extract and alkaloids were evaluated in promastigotes of L. amazonensis and epimastigotes of T. cruzi (Y strain).For the assay, epimastigote forms of T. cruzi (Y strain) were harvested during the exponential phase of growth, resuspended in liver infusion tryptose broth supplemented with 10% inactivated foetal bovine serum (Gibco Invitrogen Corporation, New York, NY, USA) and plated on 24-well plates at a concentration of 1 × 10 6 cells per mL.One millilitre of diluted compound was included in each well and incubated for 96 h at 28 °C.Cell density was determined by counting the parasites in a hemocytometer chamber (Improved Double Neubauer) under a light microscope.All assays were performed in duplicate on separate occasions.The results were expressed as percentage of inhibition in relation to the control.The 50% inhibitory concentration (IC 50 ) was determined by logarithm regression analysis of the obtained data.Parasites were treated with different concentrations (10.0, 50.0, 100.0, 500.0 and 1000.0 µg mL -1 ) of crude extract and 1.0, 5.0, 10.0, 50.0 or 100.0 µg mL -1 of isolated alkaloids (4 and 5).
2 and 4.8 Hz) and H-6b (d H 3.01, m) to C-7 (d C 106.0) and C-2 (d C 130.5) indicated the presence of a tetrahydro-b-carboline moiety in the structure of alkaloid 2.
1Number of hydrogens bound to carbon atoms deduced by comparative analyses of { 1 H}-and DEPT-13 C NMR spectra.Assignments based on1

Table 3 .
Effect of the extracts and alkaloids isolated from P. prunifolia against promastigotes of L. amazonensis and epimastigotes of T. cruzi Values represent the mean ± S.D. of at least three experiments.