Antiparasitic Activities of Acridone Alkaloids from Swinglea glutinosa ( Bl . ) Merr

Onze alcalóides acridônicos isolados de Swinglea glutinosa (Bl.) Merr. foram avaliados para suas atividades in vitro contra linhagens de Plasmodium falciparum sensíveis a cloroquina 3D7, Trypanosoma brucei rhodesiense STIB9000 e Leishmania donovani L82. Ensaios com células KB foram também executados com o objetivo de se determinar o grau de toxicidade das substâncias ativas contra os parasitas. Nove dos compostos apresentaram IC 50 entre 0,3 e 11,6 μM contra P. falciparum. Em contraste, um pequeno número de compostos mostrou atividade significativa contra T. brucei rhodesiense e nenhum apresentou atividade contra L. donovani. Entre os alcalóides três tiveram IC 50 < 1,0 μM contra P. falciparum, enquanto que contra T. b. rhodesiense cinco mostraram IC 50 < 10 μM. A caracterização dos alcalóides, 1,3,5-triidróxi-4-metóxi-10-metil-2,8bis(3-metilbut-2-enil)acridin-9(10H)-ona (1), 2,3-diidro-4,9-diidróxi-2-(2-hidróxipropan-2-il)11-metóxi-10-metilfuro[3,2-b]acridin-5(10H)-ona (2) e 3,4-diidro-3,5,8-triidróxi-6-metóxi-2,2,7trimetil-2H-pirano[2,3-a]acridin-12(7H)-ona (3), é aqui discutida. Discute-se também a relação estrutura-atividade para todos os compostos ensaiados. O isolamento e dados espectrais para os alcalóides 1-3 estão sendo aqui descritos pela primeira vez, embora em trabalho anterior tenham sido relatadas as suas atividades citotóxicas.


Introduction
Parasitic protozoa are the causative agents of human and livestock diseases infecting hundreds of millions of people every year and are collectively one of most important causes of human misery. 1 Human African trypanosomiasis (HAT), or sleeping sickness, malaria, Chagas' disease and leishmaniasis are major health problems in many countries.HAT promoted by Trypanosoma brucei rhodesiense and T. b. gambiense is endemic in over 30 African countries threatening over 60 million people.HAT has reached epidemic proportions in some countries, such as Angola, southern Sudan, Uganda and the Democratic Republic of Congo. 2,3Malaria has re-emerged as a major public health problem over the past three decades mainly because of the development of worldwide resistance of Plasmodium falciparum to chloroquine, a drug which formed the basis for cheap and effective treatment and for prophylaxis of this disease. 4Each year, approximately 300 to 500 million malaria infections lead to over one million deaths.In many endemic countries, malaria is responsible for economic stagnation, lowering the annual economic growth in some regions by up to 1.5%. 5,6Leishmaniasis is a disease caused by protozoa of the genus Leishmania.According to WHO 88 countries are affected, with 350 million people at risk.90% of cases of visceral leishmaniasis occur in India, Sudan, Bangladesh and Brazil. 7Present chemotherapy for these diseases is inadequate or toxic, or becoming ineffective due to an increase in resistance. 8he family Rutaceae contains many secondary metabolites such as alkaloids, flavonoids, coumarins, limonoids and lignans with a large spectrum of biological activities. 9Studies showed that acridone alkaloids are compounds with promising activity against P. falciparum, 10,11 and also have antiviral 12 and antiproliferative effects on cancer cell lines. 13,14The Asian genera Citrus and Swinglea are members of the Rutaceae and are included in the subfamily Aurantioideae.Citrus species have been investigated and characterized by possessing acridone alkaloids.These data stimulated an investigation of Swinglea glutinosa (Bl.)Merr. in a search for lead acridones.
Compound 2 was isolated as an amorphous powder, the molecular formula C 20 H 21 NO 6 was determined on the basis of HRESIMS, exhibiting an [M+H] + peak at m/z 372.1447.The UV and IR spectra were identical to those of 1, however some differences were observed in the 1 H and 13 C NMR.In the 1 H NMR spectrum, the characteristic signal of a hydrogen-bonded hydroxy proton at d 14.42, exchangeable with D 2 O suggested the presence of a hydroxyl group.][18][19][20][21][22] The 1 H NMR spectrum of 2 showed an ABX type aromatic spin system at d 7.15 (1 H, t, J 7.8Hz, H-7), 7.29 (1H, dd, J 7.8, 1.4Hz, H-8) and 7.80 (1H, dd, J 7.8, 1.4Hz, H-6), this last proton being deshielded by the 5-carbonyl group.The spectrum also showed one N-methyl group at d 3.85 and one O-methyl group at d 3.89.The presence of a hydroxyisopropyldihydrofuran moiety was suggested by an oxymethine proton at d 4.88 (1H, dd, J 9.4, 7.8Hz, H-2), methylene protons as two dd in an AB system at d 3.20 (1H, dd, J 15.5, 7.8Hz, H-3a) and 3.26 (1H, dd, J 15.5, 9.4Hz, H-3b), two methyl groups at d 1.33 (3H, s, H-2') and 1.29 (3H, s, H-3').The signal at d 93.0 and 71.5 in the 13 C NMR spectrum supported the presence of this substituent in the acridone nucleus. 23 2 shows 1 H, 13 C NMR data for 2 and the correlations observed in the HMBC.
Compound 3 was isolated as an amorphous powder, the molecular formula was determined as C 20 H 21 NO 6 by HRESIMS, showing an [M + H] + peak at m/z 372.1447.
The eleven acridone alkaloids isolated from S. glutinosa were tested for in vitro activity against P. falciparum, T. b. rhodesiense and L. donovani.An assay with KB cells indicated in vitro cytotoxicity.The results are summarized in Table 4.To facilitate the discussion, IC 50 values were assigned as IC 50 T,P,K against T. b. rhodesiense, P. falciparum and KB cells, respectively.
Nine out of the eleven acridone alkaloids showed IC 50 P below 10 µM, four showed IC 50 T below 10 µM and none displayed significant activity against L. donovani.
Related acridone alkaloids from Thamnosma rodesica (Bak.F.), showed activity against promastigote and amastigote forms of Leishmania major.These alkaloids have a methyl 2,3-dihydroxypropanoate chain at C-3 and C-4, 23 indicating that the substitution in the acridone skeleton is important for activity in this class of compounds.According to the data, compound 5, with one prenyl group at C-2, was the most active against P. falciparum with IC P 50 0.3 µM.Comparison of 5, 1 (IC P 50 2.6 µM) and 4 (IC P 50 2.6 µM) indicates that the second prenyl at C-8 was responsible for reducing the activity of this series.This fact was also observed by Weniger et al., 10 who performed the assay with four alkaloids from S. glutinosa on a Nigerian chloroquine-sensitive strain of P. falciparum.Analysis of the results for compounds 2, 3, 9, 10 and 11 suggests that the presence of a pyran ring is important for activity against P. falciparum and the position of this group, angular pyrano[2,3-c] (9) or linear pyrano[3,2-b] (10), did not alter the results of IC P 50 .The activity against P. falciparum observed for 6, 7 and 8, IC P 50 8.9, 29.9 and 6.1 µM, respectively, shows that presence of an O-methyl group at C-2 improves the activity.
From the 11 alkaloids tested against T. b. rhodesiense, compound 9 was the most active with an IC T 50 1.0 µM.Alkaloids 1-3 had their citotoxicity to cancer cells described in an earlier paper, 30 however here we disclose for the first time their isolation, spectral data and structure elucidation.

General
Optical rotations were measured using a Perkin Elmer polarimeter.IR spectra were recorded on a Bomem M-B Series spectrophotometer.UV absorptions were recorded using a Varian 500 SCAN UV-Vis-NIR spectrophotometer. 1 H and 13 C NMR data were recorded on Bruker ARX-200 and Bruker DRX-400 spectrometers.Spectra were recorded in acetone-d 6 and DMSO-d 6 with TMS as internal standard.All 2D NMR data were recorded at 400MHz (Bruker DRX-400), HSQC J 145 Hz; HMBC J 8 Hz.HR-MS data were recorded on a Micromass Q-Tof (QqTOF) spectrometer; column chromatography was on silica gel 60 (Merck) and Sephadex LH-20 (Pharmacia).Preparative HPLC was performed on a Shodex Asahipak GS-310 2G column.TLC was carried out using Merck aluminum-backed silica gel 60 F 254 .

Plant material
Leaves, stem and root bark were collected in Campinas (SP) at the Instituto Agronômico de Campinas, and dried in the shade.The plant was identified by Prof. Dr. Maria Inês Salgado.A voucher specimen is deposited at the Herbarium of the Departamento de Botânica of the Universidade Federal de São Carlos (HUFSCar) as number 7110.

Biological assays
Stock solutions of the compounds, plus control drugs, were prepared at a concentration of 20 mg mL -1 in DMSO (Sigma, UK), and diluted to appropriate concentrations prior to assays.IC 50 values were calculated with MSXLFIT (IDBS, UK).

P. falciparum
Chloroquine-sensitive P. falciparum strain 3D7 was maintained in human A + erythocytes in RPMI 1640 medium (Sigma, UK) supplemented with Albumax II at 37 ºC in a 5% CO 2 -air mixture.Asynchronous (65-75% ring stage) of P. falciparum intraerythrocytic cultures were set up as above, with 1% parasitemia, 2.5% hematocrit, in triplicate in 100 µL of medium in 96 well, flat-bottomed Microtest III tissue plates.Drugs were added in a threefold dilution series and cultures incubated for a total of 48 h at 37 ºC in a 5% CO 2 -air mixture.After 24 h, { 3 H} hypoxanthine (0.2 mCi) was added to each well. 25,26At the end of the assay, plates were rapidly freeze-thawed, harvested using a Tomtec Mach III cell harvester (Tomtec, CT) onto a 96well format filtermat and Meltilex TM solid scintillant (both Wallac, Finland) added prior to reading in a Microbeta 1450 scintillation counter (Wallac, Finland) at 1 min per well.

T. brucei rhodesiense
T. b. rhodesiense STIB900 bloodstream form trypomastigotes were maintained in HMI-18 medium, 27 with 15% heat-inactivated fetal calf serum (Harlan Sera Lab, UK) at 37 °C in a 5% CO 2 -air mixture.Prior to drugging, trypomastigotes were washed and resuspended in fresh medium at a concentration 2 × 10 5 trypanosoma/mL and 100 µL of this suspension was added to the drug dilutions.The top concentration for the test compounds was 30 mg mL -1 .Pentamidine was included as the standard drug.Plates were incubated for 72 h at 37 ºC in a 5% CO 2air mixture. 28At 72 h AlamarBlue was added to the plates.Plates were read after 4-5 h on a Gemini fluorescent plate reader (Sofimax Pro.3.1.1,Molecular Devices, UK) at EX/EM 530/585 nm with a filter cut-off at 550 nm.

L. donovani
L. donovani L82 amastigotes were harvested from an infected hamster (Mesocricetus auratus) spleen and used to infect murine peritoneal exudate macrophages (PEM) at a ratio of 7:1.In brief, infected cells were exposed to drug for a total of 5 days. 29The percentage of infected cells was evaluated microscopically and the percentage inhibition in comparison with untreated controls was calculated.

Cytotoxicity assays
96-well plates were seeded with KB cells at 4 × 10 4 mL -1 (100 µL per well).Drugs at 300, 30, 3 and 0.3 µg mL -1 were added in fresh overlay after 24 h, in triplicate at each concentration.Plates were incubated for 72 h at 37 °C in a 5% CO 2 -air mixture.At 72 h AlamarBlue was added to the plates.Plates were read after 4-5 h on a Gemini fluorescent plate reader (Sofimax Pro.3.1.1,Molecular Devices, UK) at EX/EM 530/585 nm with a filter cut-off at 550 nm.IC 50 values were calculated against the blanks and control samples.
0Hz, H-5'' Z)] were observed, which suggested the presence of two prenyl groups (3-methylbut-2-enyl). Their positions were confirmed at C-2 and C-8 by HMBC.The position of a prenyl group at C-2 was confirmed by the correlation of the methylene protons at d 3.40 (H-1') with C-1 (d 158.1),C-2 (d 109.4),C-3 (d 156.1),C-2' (d 123.6) and C-3' (d 131.4) in the HMBC spectrum.In addition, the correlation of 1-OH at d 14.42 with C-1 and C-9a (d 107.6) confirmed that the first prenyl group was attached to C-2.The position of the second prenyl group was confirmed by the correlation of the deshielded methylene protons at d 4.01 (H-1'') with C-7 (d 125.4),C-8 (d 135.0),C-2'' (d 125.4) and C-3'' (d 131.2), confirming that the second prenyl group was attached to C-8.The N-methyl protons showed correlation with C-4a and C-5a at d 140.2 and 139.4,respectively.The O-methyl protons showed correlation with C-4 at d 128.8.

Table 3 .
13 and13C NMR and HMBC data for compound 3 (acetone-d 6 ) a Recorded at 100 MHz; b Recorded at 400 MHz; * Assignments may be exchanged.

Table 4 .
In vitro activity against P. falciparum 3D7, T. b. rhodesiense STIB 900, L. donovani L82 and KB cells a a The mean IC 50 values of the test compounds and standard drug (n = 3, ±, σ, n = number of tests performed in three series).Drugs used as positive control: b Plasmodium falciparum; c T. brucei rhodesiense; d Leishmania donovani and e toxicity.