1 Instituto de Química, Biological Chemistry Laboratory, Universidade Estadual de Campinas, Campinas, C.P. 6154, CEP 13083-970, SP; 2 Department of Biochemistry and Immunology, Faculdade de Medicina de Ribeirão, Universidade de São Paulo, Ribeirão Preto, SP; 3 Instituto Adolfo Lutz, Ribeirão Preto, SP; 4 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP; 5 Department of Immunology, IOC, Fundação Oswaldo Cruz, RJ; 6 Department of Ultra-structure and Cellular Biology, IOC, Fundação Oswaldo Cruz, RJ; 7 NCA, Univesidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil
Received 6 August 2001; returned 20 April 2002; revised 10 May 2002; accepted 25 July 2002
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Abstract |
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Introduction |
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Chagas disease, which is caused by the parasite Trypanosoma cruzi, is endemic in Latin America, affecting 1618 million people, and is a threat to 100 million people who are at risk of infection mainly in rural areas or via blood transfusion.3 The life cycle of T. cruzi also involves a vertebrate host and an invertebrate host. The trypomastigote form ingested by the insect differentiates into an epimastigote, which in the posterior intestine differentiates into a metacyclic trypomastigote. This latter form invades vertebrate cells and undergoes differentiation into an amastigote, which after several reproductive cycles transforms into trypomastigotes.
Besides the serious problems with Chagas disease, there has been a dramatic increase in the number of cases of leishmaniasis and tuberculosis, as well as other opportunistic infections, in human immunodeficiency virus (HIV)-infected patients.4,5 In addition, the development of drug resistance by the pathogens6 has aggravated public health risks and new chemotherapeutic agents are urgently needed.
The chemotherapy of Chagas disease is still inadequate. Nowadays, the only available therapeutic agent in Latin America is the nitroheterocycle benznidazole (N-benzyl-1,2-nitro-1-imidazole-acetamide), which has variable efficacy and severe side effects. Since 1984, the World Health Organization has recommended the use of Crystal Violet in blood banks in endemic areas to prevent the transmission of Chagas disease by blood transfusion. Although Crystal Violet presents no substantial side effects, blood micro-agglutination and potential mutagenicity have been reported,7 and the bluish colour conferred upon blood and tissues is not accepted well by the patients. In this context, intensive research has been directed at finding alternative drugs to both benznidazole and Crystal Violet.8,9
Anti-protozoal activity has been observed in a wide range of nitroheterocyclic compounds, including nitrothiazoles, nitroimidazoles and nitrofurans. Also, other synthetic compounds and plant extracts have been identified as possible agents against Leishmania, Trypanosoma or Plasmodium, but their potential usefulness is limited by their cytotoxicity and low bioavailability.10
Leishmaniasis is a significant cause of morbidity and mortality in several countries of the world, affecting more than 12 million people. Leishmania amazonensis is found in different regions in Brazil and is responsible for various forms of the disease, including cutaneous, hyperergic mucocutaneous, anergic diffuse cutaneous and visceral leishmaniasis.11 This disease is transmitted to the vertebrate host by the bite of a sandfly, the promastigote being the infective form of the parasite, which penetrates a mononuclear phagocyte, differentiates into an amastigote and proliferates intracellularly.
The treatment of leishmaniasis involves the administration of pentavalent antimonials (sodium stibogluconate, Pentostam or meglumine, Glucantine), pentamidine or amphotericin B. These drugs are potentially toxic,12 although this is less of a problem with liposomal amphotericin B.
We have previously demonstrated the trypanocidal, antimycobacterial and leishmaniasis activity of 3-[4'-bromo-(1,1'-biphenyl)-4-yl]-N,N-dimethyl-3-(4-X-phenyl)-2-propen-1-amine derivatives.1317 In the present study, a new N,N-dimethyl-2-propen-1-amine derivative was synthesized and its biological activity was evaluated against extracellular mycobacterial strains, the three life cycle forms of T. cruzi and promastigotes of L. amazonensis. The structure of this new compound is characterized by a biphenyl ring, a lateral chain of N,N-dimethyl-2-propen-1-amine and a thienyl ring, which probably contributes to its biological activity.
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Materials and methods |
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The chemicals used in this study were purchased from Aldrich-Sigma or Merck and were used without further purification, except the solvent tetrahydrofuran (THF), which was treated with anhydrous calcium hydride, refluxed with metallic sodium and distilled.
Synthesis
Through a FriedelCrafts reaction, the ketone 4'-bromo-(1,1'-biphenyl)-4-yl 2-thienylmethanone (III) was obtained as an intermediate for the synthesis of compound IV (Figure 1) following a Wittig reaction as previously described.17
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Compound III. 49-bromo-[1,19-biphenyl]-4-yl 2-thienylmethanone. To a mixture of 4-bromobiphenyl (I) (4.11 mmol), AlCl3 (3 mmol) and CS2 (15 mL), 2-thiophenecarbonyl chloride (II) (3.74 mmol) was added dropwise (0.40 mL) (Figure 1). The reaction mixture was refluxed for 8 h and cooled to room temperature by the addition of crushed ice. The solvent was distilled and the reaction product was extracted with CH2Cl2 (100 mL) and treated with a 10% NaOH solution (20 mL). The organic layer was separated, dried over MgSO4, filtered and evaporated under reduced pressure to give the solid crystalline product III. The purification was performed through re-crystallization with CH2Cl2/hexane.
Compound IV. 3-[49-bromo-(1,19-biphenyl)-4-yl]-N,N-dimethyl-3-(2-thienyl)-2-propen-1-amine. N-butyllithium (6.42 mmol) in a hexane solution (2.5 mmol/L) was added slowly to a flask containing ß-dimethylamino ethyl triphenyl phosphonium bromide (2.92 mmol) and 40 mL of THF at 0°C with stirring in an argon atmosphere. After 30 min, the ketone III (1.46 mmol) was added and the reaction system kept overnight at room temperature under agitation. The reaction was stopped by the addition of water and product IV was extracted with CH2Cl2 and purified in two steps by percolation in a silica gel column using ethyl acetate/CH3OH 010% as eluent, and by thin layer chromatography (TLC) with ethyl acetate/CH3OH 10% as eluent.
Chemistry
The reactions were monitored by TLC on UV-254 and the purity of the compounds determined. Mass spectra (MS) were taken on a spectrometer VG Autospec under electron impact at an ionization irradiation energy of 70 eV. The ratios m/z and the relative intensities corresponded to the calculated molecular weights of the compounds prepared. 1H-NMR spectra were recorded on a Varian Gemini apparatus at a working frequency of 300 MHz in deuterated chloroform (CDCl3) using tetramethylsilane (TMS) as an internal standard (chemical shifts in , ppm). Analytical TLC was carried out on pre-coated plates (silica gel 60 F254) and the spots were visualized with UV light and in an I2 chamber. Infrared (IR) spectra (KBr) were recorded on a WinBomem spectrophotometer (Vmax in cm1) and the melting point with a Quimis apparatus. The compounds were characterized by the above-mentioned techniques and the data are listed below.
Compound III. IR (KBr, cm1) 1620 (, C=O), 1265 (
, CN). 1H-NMR (500 MHz; CDCl3/TMS,
): 7.997.18 (m, 11H, Ar). MS (m/z) 344/342 (M+·, 100), 261/259 (21), 152 (36), 111 (82). Melting point: 106120°C. Yield: 80%.
Compound IV. IR (KBr, cm1) 1480 (, SAr), 1265 (
, CN). 1H-NMR (500 MHz; CDCl3/TMS,
): 7.606.66 (m, 11H, Ar), 6.28 (1t, 1H, CH E/Z, J = 6.96 Hz); 6.23 (1t, 1H, CH E/Z, J = 6.72 Hz); 3.26 (1d, 2H, CH2 E/Z, J = 6.59 Hz); 3.01 (1d, 2H, CH2 E/Z, J = 7.08 Hz); 2.32 [1s, 6H, CH3 E/Z N(CH3)2]; 2.26 [1s, 6H, CH3 E/Z N(CH3)2]. MS (m/z) 399/397 (M+·, 100), 394/382 (39), 314/312 (8), 166 (34), 97 (39). Yield: 43%.
Cytotoxicity assays
The cytotoxic effect of compounds III and IV expressed as cell viability was assayed on the mammalian lineage Chinese hamster V79 lung fibroblasts. The cells were grown as monolayers in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 IU/mL penicillin and 100 mg/L streptomycin in a humidified incubator with 5% CO2 in air at 37°C. Stock solutions of compounds III and IV were prepared in DMSO and diluted in DMEM. The final concentration of the solvent in the assay was 0.4%. The V79 cells were plated at a density of 3 x 104 cells/mL in 96-well plates and after 48 h the compounds were added in concentrations ranging from 5 to 350 µmol/L. The controls received no drugs and each drug concentration was tested in eight replicates and repeated three times in separate experiments. After 24 h of incubation, three independent endpoints for cytotoxicity were evaluated: nucleic acid content (NAC), reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Neutral Red uptake (NRU).
NAC. Cell numbers in control and treated wells were estimated from their total NAC according to Cingi et al.18 The cells were washed twice with cold PBS and a soluble nucleotide pool was extracted with cold ethanol. The cell monolayers were then dissolved in 0.5 mol/L NaOH and incubated at 37°C for 1 h and the absorbance read at 260 nm. Results are expressed by comparing the absorbance of treated cells and controls.
MTT. The tetrazolium reduction assay was performed according to the method of Denizot & Lang.19 Briefly, the cells were washed once with PBS and 0.1 mL of serum-free medium containing MTT (1 mg/mL) was added to each well. After incubation for 4 h, the supernatant was removed and the blue formazan product obtained was dissolved in 0.1 mL of ethanol with stirring for 15 min on a microtitre plate shaker and the absorbance was read at 570 nm (Elisa reader Metertech Inc. model 960).
NRU. The NRU assay was performed according to the method of Borenfreund & Puerner.20 After 4 h of incubation with serum-free medium containing Neutral Red (50 mg/L), the cells were washed quickly with PBS and then 0.1 mL of acetic acid at 1% (v/v) in a solution of ethanol 50% (v/v) was added to each well to extract the dye. After rapid agitation on a microtitre plate shaker, the absorbance was read at 540 nm.
Antimicrobial susceptibility testing
The following strains were used: Mycobacterium tuberculosis H37Rv ATCC 27294, M. tuberculosis H37Ra ATCC 25177, M. avium ATCC 15769, M. kansasii ATCC 12478, M. intracellulare ATCC 25169 and M. malmoense ATCC 29571. Stock solutions of compounds III and IV were prepared in dimethylsulfoxide (DMSO; Sigma Chemical Co., St Louis, MO, USA). The mycobacteria were subcultured on LowensteinJensen medium at 37°C for 3 weeks, followed by subculture in Middlebrook 7H9 broth medium at 37°C for at least 10 days, until bacterial density corresponding to a 1.0 McFarland turbidity standard was reached. The tests were performed through the microplate Alamar Blue assay as previously reported.21 Briefly, the mycobacteria suspensions were diluted 1:25 in Middlebrook 7H9 broth medium (4 x 105 mycobacteria/mL) and 100 µL was added to 96-well microplates containing 100 µL of serial dilutions of compound III or IV in the same medium (1640 µmol/L). After incubation at 37°C for 6 days, 25 µL of a 1:1 (v/v) mixture of Alamar Blue reagent and 10% Tween 80 was added and the plates were re-incubated at 37°C for 24 h. A change in the colour from blue to pink was observed in the wells where the mycobacteria grew. The visual MICs were defined as the lowest drug concentration that prevented the colour change.
Trypanocidal assays
The Y strain of T. cruzi was used.22 Trypomastigote forms were obtained at the peak of parasitaemia from the blood of infected albino mice, isolated by differential centrifugation. Epimastigote forms were maintained in liver infusion tryptose (LIT) medium and harvested during the exponential phase of growth. Amastigote forms were collected from the supernatant of trypomastigote-infected J-774G-8 macrophage cultures.23 A stock solution of compound IV was prepared in DMSO (Sigma Chemical Co.), and diluted in the appropriate medium (see below) in the concentration range 0.5500 µmol/L, with the final concentration of the solvent in the experiments never exceeding 0.1%.
Trypomastigotes were resuspended with DMEM to a parasite concentration of 1 x 107 cells/mL in the absence or presence of 10% of blood. The parasite suspension (100 µL) was added to the same volume of a solution of compound IV, previously prepared at twice the desired concentration also in DMEM in 96-well plates, and then incubated at 4 or 37°C. Untreated and Crystal Violet-treated parasites were used as controls.24 A similar procedure was employed in the assays with the proliferative forms of T. cruzi, and the incubation temperature was 28°C.25 In the assays with amastigotes, the medium employed was DMEM plus 10% heat-inactivated FCS. For epimastigotes, LIT medium was used.
Cell counts were performed after 24 h of incubation, and the activity of compound IV was expressed as ED50/24 h, corresponding to the concentration that leads to 50% parasite lysis. The motility and morphology of the parasites were monitored by optical microscopy (Zeiss, Axioplan, Oberköchen, Germany).
Leishmanicidal assays
The assays were performed with promastigote forms of L. amazonensis (strain MHOM/BR/77/LTB0016) and the characterization of the strain was performed by molecular techniques such as isoenzyme electrophoresis and indirect radio-immunoassay using specific monoclonal antibodies.26 The parasites were maintained at 26°C in Schneiders Drosophila medium pH 7.2 (Gibco, Paisley, UK) supplemented with 10% FCS. A stock solution of compound IV was prepared in DMSO, and the compound was diluted in Schneiders Drosophila medium, with the final concentration of the solvent in the experiments never exceeding 1.4%.
The parasites were harvested in the late log phase, re-suspended in the same medium (to keep promastigotes in the same growth phase), and the concentration adjusted to 4 x 106 cells/mL. They were then incubated at 26°C with compound IV in concentrations ranging from 0.4 to 804 µmol/L. After 24 h of incubation, the parasites were counted in a Neubauer chamber and compared with controls that had DMSO plus parasites and parasites alone. The results were expressed as ED50/24 h. All tests were carried out in triplicate and pentamidine isethionate (May & Baker Lab., London, UK) was used as the reference drug.
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Results and discussion |
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The compounds were synthesized by the classical FriedelCrafts reaction leading to ketone III, followed by a Wittig reaction to synthesize compound IV. The physical data of compounds III and IV are shown in Materials and methods, and their structures in Figure 1.
The IR spectrum of compound III showed a band at 1620 cm1 attributed to the carbonyl group. The hydrogens of the phenyl and thienyl rings showed a multiplet sign at 7.997.18 by 1H-NMR. In the case of compound IV, the 1H-NMR spectrum showed, besides the multiplet signal of the aromatic hydrogens (
7.66.66), two triplets (
6.28 and 6.23) due to the CH groups and two doublets (3.26 and 3.01) due to the CH2 groups. These double doublets and triplets are characteristic of the presence of the geometric isomers E and Z in these molecules, as demonstrated in previous work of our group with another N,N-dimethyl-2-propen-1-amine derivative.17 By MS, the molecular ions of 344/342 and 399/397 were detected in compounds III and IV, respectively. The structures of both compounds (Figure 1), obtained with a high degree of purity, are in agreement with the physical and spectral data.
Biological assays
Toxicity screening tests are routinely used in drug development programmes to determine whether the pharmacological concentration desired also induces toxic effects. The viability assays with V79 cells, which measure the cytotoxicity of the synthesized compounds, were applied in a broad range of concentrations. The cytotoxic effects were measured by NAC, MTT and NRU techniques. Figures 2 and 3 show, respectively, the dose-dependent effect of compounds III and IV on the mammalian cells, and Table 1 states the inhibitory concentrations (ICs) achieved after plotting the percentage of cytotoxicity versus the concentration of the drug. By the MTT assay, incubation for 24 h with 350 µmol/L compound III, 90% of the cells remained viable, indicating that this intermediate of the synthesis of compound IV is non-toxic to V79 cells. The effect of compound III on lysosomes (NRU) and cellular macromolecules (NAC) is also moderate, since at the same concentration the viability of the cells was >50% (Table 1). On the other hand, compound IV exhibited toxicity to V79 cells with IC50/24 h of 23.9, 39.8 and 28.2 µmol/L determined, respectively, by the NAC, MTT and NRU tests. Both compounds III and IV caused an oxidative stress on V79 cells, as indicated by the increase in cell viability at 50 and 10 µmol/L, respectively (see Figures 2 and 3). The internal environment of proliferating cells is more reduced than that of non-proliferating ones. Specifically, the ratios of NADPH/NADP, FADH/FAD, FMNH/FMN and NADH/NAD increase during proliferation. The reduction of MTT involves the acceptance of electrons from NADPH, FADH, FMNH or NADH, which are present in high concentrations during cell proliferation.
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The susceptibility patterns of the mycobacterial species to compound IV and compounds with standard antituberculosis activities are displayed in Table 2. One week after incubation, the results from visually determined MICs were obtained using Alamar Blue. This dye is a general indicator of cellular growth and/or viability; the blue, non-fluorescent, oxidized form becomes pink and fluorescent upon reduction. M. tuberculosis H37Rv and H37Ra were more susceptible to compound IV, although just by one dilution, than M. avium, M. kansasii or M. malmoense.
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The mechanism of action of compound IV is unknown, but based on its structure and previous work with benzo[b]-thiophene-4,7-quinones,41,42 the thiophene nucleus (thienyl ring) plays an important role in antiprotozoal and antifungal activity.43 Several compounds with a thienyl ring in their structure are responsible for antimycotic activity;4446 the thiophene nucleus, possibly being a mimetic of imidazole or triazole, binds to the iron atom of the cytochrome P450 haem and thereby displays potent antimycotic activity.46 Although structureactivity relationship studies showed that substitution of the phenyl ring by a thienyl ring contributed to an increase in the antimycotic activity,6 in our study this substitution decreased the leishmanicidal (ED50/24 h increased from 0.6283 to 3.0 ± 0.3 µmol/L),15 trypanocidal (ED50/24 h increased from 18.8 ± 1.2 to 60.6 ± 6.8 µmol/L)17 and antimycobacterial activities (MIC increased from 10 to 20 µmol/L).14 The phenyl and thienyl rings have a log P of 2.13 and 1.81, respectively, whereas the resonance energies are 36 and 29 kcal/mol, respectively. These characteristics are probably important for the kind of activity discussed here.
In summary, compound IV showed an excellent antileishmanial activity and showed some antituberculosis activity.
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Acknowledgements |
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Footnotes |
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References |
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2 . Gbayisomore, A., Lardizabal, A. A. & Reichman, L. B. (2000). Update: prevention and treatment of tuberculosis. Current Opinion in Infectious Disease 13, 1559.[ISI][Medline]
3 . World Health Organization. (1998). Chagas disease. Weekly Epidemiology Records 1/2, 1.
4 . Albrecht, H., Sobottka, I., Emminger, C., Jablonowski, H., Just, G., Stoehr, A. et al. (1996). Visceral leishmaniasis emerging as an important opportunistic infection in HIV-infected persons living in areas nonendemic for Leishmania donovani. Archives of Pathology and Laboratory Medicine 120, 18998.[Medline]
5 . Badri, M., Ehrlich, R., Wood, R., Pulerwitz, T. & Maartens, G. (2001). Association between tuberculosis and HIV disease progression in a high tuberculosis prevalence area. International Journal of Tuberculosis and Lung Disease 5, 22532.[ISI][Medline]
6
.
Espinal, M. A., Laszlo, A., Simonsen, L., Boulahbal, F., Kim, S. J., Reniero, A. et al. (2001). Global trends in resistance to antituberculosis drugs. New England Journal of Medicine 344, 1294303.
7 . Thomas, S. M. & McPhee, D. G. (1984). Crystal violet: a direct-acting frameshift mutagen whose mutagenicity is enhanced by mammalian metabolism. Mutation Research 140, 1657.[ISI][Medline]
8 . De Castro, S. L. (1993). The challenge of Chagas disease chemotherapy: an update of drugs assayed against Trypanosoma cruzi. Acta Tropica 53, 8398.[ISI][Medline]
9 . Urbina, J. A. (1999). Chemotherapy of Chagas disease: the how and the why. Journal of Molecular Medicine 77, 3328.[ISI][Medline]
10 . Croft, S. L. (1997). Pharmacological approaches to antitrypanosomal chemotherapy. Memórias do Instituto Oswaldo Cruz 94, 21520.
11 . Leon, L. L., Machado, G. M. C., Barral, A., Carvalho-Paes, L. E. & Grimaldi, G., Jr (1992). Antigenic differences among Leishmania amazonensis isolates and their relationship with the clinical forms of the disease. Memórias do Instituto Oswaldo Cruz 87, 22934.
12 . Olliaro, P. L. & Bryceson, A. D. M. (1993). Practical progress and new drugs for changing patterns of leishmaniasis. Parasitology Today 9, 3238.[ISI]
13 . Pereira, D. G., De Castro, S. L. & Durán, N. (1998). Activity of N,N-dimethyl-12-propen-1-amine derivatives in mice experimentally infected with Trypanosoma cruzi. Acta Tropica 69, 20511.[ISI][Medline]
14 . De Souza, A. O., Santos Júnior, R. R., Ferreira-Júlio, J. F., Rodriguez, J. A., Melo, P. S., Haun, M. et al. (2001). Synthesis, antimycobacterial activities and cytotoxicity on V79 of 3-(4'-bromo1,1'-biphenyl-4-yl)-3-(4-X-phenyl)-N,N-dimethyl-2-propen-1-amine derivatives. European Journal of Medicinal Chemistry 36, 84350.
15 . De Souza, A. O. & Durán, N. (1998). Synthesis and antileishmaniasis activities of N,N-dimethyl-2-propen-1-amine derivatives. Brazilian Patent PIBr. 9902748-8.
16
.
De Souza, A. O., Sato, D. N., Aily, D. C. G. & Durán, N. (1998). In vitro activity of N,N-dimethyl-2-propen-1-amines against Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 42, 4078.
17 . De Conti, R., Gimenez, S. M. N., Haun, M., Pilli, R. A., De Castro, S. L. & Durán, N. (1996). Synthesis and biological activities of N,N-dimethyl-2-propen-1-amine derivatives. European Journal of Medicinal Chemistry 31, 14.
18 . Cingi, M. R., De Angelis, I., Fortunati, E., Reggiani, D., Bianchi, V., Tiozzo, R. et al. (1991). Choice and standardization of test protocols in cytotoxicology: a multicentre approach. Toxicology In Vitro 5, 11925.[ISI]
19 . Denizot, F. & Lang, R. (1986). Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of Immunological Methods 89, 2717.[ISI][Medline]
20 . Borenfreund, E. & Puerner, J. A. (1984). A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/VN 90). Journal of Tissue Culture Methods 9, 79.
21 . Collins, L. A. & Franzblau, S. G. (1997). Microplate Alamar Blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrobial Agents and Chemotherapy 41, 10049.[Abstract]
22 . Silva, L. H. P. & Nussenszweig, V. (1953). Sobre uma cepa de Trypanosoma cruzi virulenta para o camundongo branco. Folia Clinica Biologica 20, 191207.
23 . De Castro, S. L., Meirelles, M. N. L. & Oliveira, M. M. (1987). Trypanosoma cruzi: adrenergic modulation of cAMP role in proliferation and differentiation of amastigotes in vitro. Experimental Parasitology 64, 36875.[ISI][Medline]
24 . De Castro, S. L., Pinto, M. C. F. R. & Pinto, A. V. (1994). Screening of natural and synthetic drugs against Trypanosoma cruzi. 1Establishing a structure/activity relationship. Microbios 78, 8390.[ISI][Medline]
25 . De Castro, S. L., Soeiro, M. N. C., Higashi, K. O. & Meirelles, M. N. L. (1993). Differential effect of amphotericin B on the three evolutive stages of Trypanosoma cruzi and on the host cellparasite interaction. Brazilian Journal of Medical and Biological Research 26, 121929.[ISI][Medline]
26 . Grimaldi, G., Jr, David, J. R. & McMahon-Pratt, D. (1987). Identification and distribution of New World Leishmania species characterized by serodeme analysis using monoclonal antibodies. American Journal of Tropical Medicine and Hygiene 36, 27087.[ISI][Medline]
27 . De Reuck, A. V. S. & Cameron, M. P. (1963). The reversible activation of lysosomes in normal cells and the effect of pathological conditions. In Lysosomes (De Reuck, A. V. S. & Cameron, M. P., Eds), pp. 362375. Little, Brown & Co., Boston, MA, USA.
28 . Schlemper, B. R., Chiari, E. & Brener, Z. (1977). Growth inhibition drug test with Trypanosoma cruzi culture forms. Journal of Protozoology 24, 5447.[ISI][Medline]
29 . De Castro, S. L., Soeiro, M. N. C. & Meirelles, M. N. L. (1992). Trypanosoma cruzi: effect of phenothiazines on the parasite and on its interaction with host cells. Memórias do Instituto Oswaldo Cruz 87, 20915.
30 . Lopes, J. N., Cruz, F. S., Do Campo, R., Vasconcellos, M. E., Sampaio, M. C. R., Pinto, A. V. et al. (1978). In vitro and in vivo evaluation of the toxicity of 1,4-naphthoquinone and 1,2-naphthoquinone derivatives against Trypanosoma cruzi. Annals of Tropical Medicine and Parasitology 72, 52331.[ISI][Medline]
31 . Rovai, L. E., Aoki, A., Gerez de Burgos, N. M. & Blanco, A. (1990). Effect of gossypol on trypomastigotes and amastigotes of Trypanosoma cruzi. Journal of Protozoology 37, 2806.[ISI][Medline]
32 . Neves-Pinto, C., Dantas, A. P., De Moura, K. C. G., Emery, F. S., Polequevitch, P. F., Pinto, M. C. F. R. et al. (2000). Chemical reactivity studies with naphthoquinones from Tabebuia with anti-trypanosomal efficacy. Arzneimittel-Forschung-Drug Research 50, 11208.
33 . Araujo, C. A. C., Alegrio, L. V. & Leon, L. L. (1998). Antileishmanial activity of compounds extracted and characterized from Centrolobium sclerophyllum. Phytochemistry 49, 7514.[ISI]
34 . Marsden, P. D., Sampaio, R. N. R., Carvalho, E. M., Veiga, J. P. T., Costa, J. L. M. & Llanoscuentas, E. A. (1985). High continuous antimony therapy in two patients with unresponsive mucosal leishmaniasis. American Journal of Tropical Medicine and Hygiene 34, 7103.[ISI][Medline]
35 . Afrin, F., Dey, T., Anam, K. & Ali, N. (2001). Leishmanicidal activity of stearylamine-bearing liposomes in vitro. Journal of Parasitology 87, 18893.[ISI][Medline]
36 . Wiese, M. & Gorckel, I. (2001). Homologues of LMPK, a mitogen-activated protein kinase from Leishmania mexicana, in different Leishmania species. Medical Microbiology and Immunology 190, 1922.[ISI][Medline]
37 . Croft, S. L. & Yardley, V. (2002). Chemotherapy of leishmaniasis. Current Pharmaceutical Design 8, 31942.[ISI][Medline]
38 . Loiseau, P. M. & Bories, C. (1999). Recent strategies for the chemotherapy of visceral leishmaniasis. Current Opinion in Infectious Diseases 12, 55964.[ISI]
39 . Escobar, P., Matu, S. & Croft, S. L. (2002). Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH3 (edelfosine) and amphotericin B. Acta Tropica 81, 1517.[ISI][Medline]
40
.
Escobar, P., Yardley, V. & Croft, S. L. (2001). Activities of hexadecylphosphocholine (miltefosine), AmBisome, and sodium stibogluconate (Pentostam) against Lerishmania donovani in immunodeficient scid mice. Antimicrobial Agents and Chemotherapy 45, 18725.
41 . Kayser, O., Kiderlen, A. F., Laatsch, H. & Croft, S. L. (2000). In vitro leishmanicidal activity of monomeric and dimeric naphthoquinones. Acta Tropica 77, 30714.[Medline]
42 . Valderrama, J., Fournet, A., Valderrama, C., Bastias, S., Astudillo, C., De Arias, A. et al. (1999). Synthesis and in vitro antiprotozoal activity of thiophene ring-containing quinones. Chemical and Pharmaceutical Bulletin 47, 12216.
43 . Ram, V. J., Goel, A., Shukla, P. K. & Kapil, A. (1997). Synthesis of thiophenes and thieno[3,2-c]pyran-4-ones as antileishmanial and antifungal agents. Bioorganic and Medicinal Chemistry Letters 7, 31016.
44 . Nussbaumer, P., Ryder, N. S. & Stutz, A. (1991). Allylamine antimycoticsrecent trends in structureactivity-relationships and syntheses. Pesticide Science 31, 43755.[ISI]
45 . Nussbaumer, P., Petranyi, G. & Stutz, A. (1991). Synthesis and structureactivity-relationships of benzo[b]thienylallylamine antimycotics. Journal of Medicinal Chemistry 34, 6573.[ISI][Medline]
46 . Stutz, A., Georgopoulos, A., Granitzer, W., Petranyi, G. & Berney, D. (1986). Synthesis and structureactivity relationships of naftifine-related allylamine antimycotics. Journal of Medicinal Chemistry 29, 11225.[ISI][Medline]
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