New Pyrone and Quinoline Alkaloid from Almeidea rubra and their Trypanocidal Activity

Alessandra R. P. Ambrozin, Jair Mafezoli , Paulo C. Vieira*, João B. Fernandes, M. Fátima das G. F. da Silva, Javier A. Ellena and Sérgio de Albuquerque c Departamento de Química, Universidade Federal de São Carlos, Rod. Washington Luiz, km 235, CP 676, 13565-905 São Carlos SP, Brazil Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador São Carlense, 400, 13560-970 São Carlos SP, Brazil


Introduction
The genus Almeidea belongs to Rutaceae family and is widely distributed in Brazil.Data about the secondary metabolites of this genus are available.From Almeidea guyanensis was isolated C-arabinosyl flavones, 2-and 4quinolones, and flavone C-glycosides. 1 We have previously reported the isolation of triterpenoids, acridone, 2-quinolone, and furoquinoline alkaloids, and a new tetrahydrofuroquinoline alkaloid from A. coeruela and A. rubra 2 besides steroids, one sesquiterpene, one cromone, and one flavone. 3agas' disease (American Trypanosomiasis) is caused by the flagellate protozoan Trypanosoma cruzi, affecting more than 18 million people in Latin America. 4Its treatment is still a challenge and new less toxic and more effective drugs are necessary.
As a part of our search for new trypanocidal compounds and our studies on the chemistry of Rutaceae, a new specimen of Almeidea rubra A. St.-Hil.was investigated.Thus, here we report the isolation, identification and characterization of two new substances (1 and 2a) and seven known compounds (3-9) from methanol extract of leaves of this plant, in addition to their trypanocidal activities.
Compound 1 was isolated as a yellow powder and its structure was determined by analysis of NMR and LRMS.
All these results allowed us to propose the structure 4methoxy-6-[2-(methylamino)phenyl]-2H-pyran-2-one for compound 1, which is described for the first time in the literature and represents a new class of compounds formed by the reaction of anthranilic acid and two acetyl/malonyl-CoA units.
Single crystal X-ray diffraction established undoubtedly the structure of 1 as a derivative of anthranilic acid (Figure 2).
The crystal structure of compound 1 shows the presence of two independent molecules per asymmetric unit (Figure 2).The main conformational difference between both independent molecules is centered in the dihedral angle between the least square planes that pass through the aromatic rings in each molecule.The value of this angle in molecule 1 is 33.58 (8)° and in molecule 2 is 34.79(8)°.The crystal structure also shows the presence of one intramolecular interaction in each molecule.These interactions are slightly different due to the conformation  changes.The analyses of the crystal packing show the presence of four intermolecular interactions of the type C-H…O that give rise to the formation of chains along the (101) direction.These chains are connected with the chain just above and below it through three intermolecular interactions of the N-H…O type.
The quinoline and acridine alkaloids, which are widely distributed in Rutaceae, arise from combination of anthranilic acid and acetate/malonate. 10In these cases, the ciclization takes place by nucleophilic attack of nitrogen atom to the carboxyl carbon.However, compound 1 should be formed by the nucleophilic attack of the oxygen atom to the carboxyl carbon, giving rise an α-pyrone.
We have previously reported the isolation of the new tetrahydrofuroquinoline alkaloid 2b (Figure 1) from A. coerulea. 2Reinvestigation of A. rubra afforded an alkaloid 2a whose spectral data seem to be very similar to those of 2b.This alkaloid was isolated as a white powder and identified by NMR and LRMS.The molecular formula C 20 H 25 NO 6 of 2a was defined by elemental analysis.
In the 1 H NMR spectrum of 2a were observed a pair of doublets (J 2.6 Hz) at δ 7.58 and 6.97, and a singlet at δ 4.30, which indicate that 2a was a tetrahydrofuroquinoline alkaloid.The signals at δ 2.78, 2.20, and 2.01, assigned to H-5, and H-6, respectively, confirmed that 2a had a reduced furoquinoline ring.Analysis of HSQC correlations and literature data 2 allowed us to attribute undoubtedly chemical shifts to methyl, methylene, vinyl, furanyl hydrogen/carbon of 2a (Table 2).
The assignments of quaternary carbons of 2a were made from observed correlations in the HMBC spectrum.The chemical shift of C-4 was deduced from 3 J correlation between methoxyl hydrogens at C-4 (δ 4.30) and one carbon at δ 158.2.The correlation of the other methoxyl hydrogens (δ 3.14) and one carbon at δ 79.7 determined  The close similarity between the chemical shifts observed for carbons of reduced furoquinoline ring when compared with the model compound 7-O-acetylhaplophyllidine (2b) 2 led us to suggest a trans relationship between the 7-acetoxy and 8-methoxy groups.Alkaloid 2a, rel-(7R,8R)-8-[(E)-3-hydroxy-3-methyl-1-butenyl]-4,8-dimethoxy-5,6,7,8-tetrahydrofuro [2,3-b]quinoline-7yl acetate, is described for the first time in the literature, and the occurrence of tetrahydrofuroquinoline alkaloid such as 2a and 2b seems to be restricted to the genera Almeidea and Haplophyllum of Rutaceae family. 2,11n order to improve the therapy of Chagas disease, we have been studying species of the order Rutales (Rutaceae, Meliaceae, Simaroubaceae, Burseraceae, and Cneoraceae) aiming to isolate hit compounds that could be used in the development of new antichagasic drugs.
Compounds described in this paper were isolated from an active fraction of A. rubra (62.9% lysis -percent reduction of the parasite number) on the tripomastigote forms of T. cruzi. 12Their in vitro trypanocidal actions are shown on Table 3.Three of them (7-9) could not be assayed due to their instability.
The results showed that all of compounds had moderate trypanocidal actions and kokusagine (6) was the most active one.Probably, they are responsible by the activity of the A. rubra fraction. 12However, they could not be considered good prototype for the development of new antichagasic drugs, mainly when their activities are compared with crystal violet and some lignans also isolated from other Rutaceae. 13

General experimental procedures
The 1 H NMR, 13 C NMR and 2D correlation spectra were obtained in CDCl 3 using either Bruker DRX-200 and ARX-400 NMR spectrometers, and using tetramethylsilane (TMS) as internal standard.

Plant material
The leaves of Almeidea rubra A. St.-Hil.were collected in May 2000 in Espírito Santo State, southeast of Brazil.The plant material was identified by Dr. José Rubens Pirani from the Department of Botany, University of São Paulo (Brazil) and the voucher herbarium specimen (Pirani et al. 4746) was deposited at the Herbarium of that Department.

Single crystal X-ray analysis
Low temperature X-ray diffraction data collection was performed at 120(2) K, on an Enraf-Nonius Kappa-CCD diffractometer equipped with an Oxford Cryosystem liquid N 2 device, using graphite-monochromated MoKα radiation (0.71073 Å).Data were collected up to 50° in 2θ, with a redundancy of 4. The final unit cell parameters were based on all reflections.Data collections were made using the COLLECT program; 14 integration and scaling of the reflections were performed with the HKL Denzo-Scalepack system of programs. 15No absorption corrections were applied.
The structure was solved by direct methods with SHELXS-97. 16The model was refined by full-matrix least squares on F 2 with SHELXL-97. 17All the hydrogen atoms were set isotropic and freely refined.The programs SHELXL-97, 17 and ORTEP-3 18 were used within WinGX 19 to prepare materials for publication.Atomic coordinates, bond lengths and angles, and thermal parameters have been deposited at the Cambridge Crystallographic Data Centre.
The supplementary crystallographic data have been sent in electronic format to the Cambridge Crystallographic Data Centre, as CIF file No. CCDC 242119.These can be obtained obtained free of charge by contacting the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; www.ccdc.cam.ac.uk/conts/retrieving.html; deposit@ccdc.cam.ac.uk).

Trypanocidal activity in vitro
As described by Ambrozin et al., 12 the bioassays were carried out using blood of infected Swiss albino mice, which was collected by cardiac puncture at the peak of parasitemic infection (7 th day of infection for Y strain of T. cruzi).The infected blood was diluted with healthy mice blood to achieve a concentration of 2.10 6 forms/mL.Solutions of the compounds were prepared by dissolving in dimethylsulfoxide (DMSO).The activities of substances were evaluated at 500, 250, and 100 µg/mL.
The bioassays were performed in triplicate on titration microplates (96 wells) which contained 400 µL mixture/ well.The plates were incubated at 4 o C, and the number of parasites counted after 24 h, following the method described by Brener. 20nfected blood with the same volume of DMSO was used as control, and gentian violet to a concentration of 250 µg/mL was used as positive control.
The activity is expressed as percent reduction of the parasite number (lysis) and IC 50 values were calculated using the program GraphPad Prims v.3.0.

Table 3 .
Trypanocidal activity of compounds isolated from Almeidea rubra