Antileishmanial activity of hydrolyzable tannins and their modulatory effects on nitric oxide and tumour necrosis factor-alpha release in macrophages in vitro.

A series of 27 hydrolyzable tannins and related compounds was tested for antiparasitic effects against both extracellular promastigote and intracellular amastigote Leishmania donovani organisms. In parallel, the compounds were evaluated for their immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and interferon (IFN)-like properties using several functional assays. Of the series of polyphenols tested, only gallic acid (54 microM NO) and its methyl ester (32 microM NO) induced murine macrophage-like RAW 264.7 cells to release NO in appreciable amounts (IFN-gamma/LPS 119 microM NO). The in vitro TNF-inducing potential of the polyphenols examined increased in the order of oligomeric ellagitannins (EC(50) > 25 microg/ml) < monomeric ellagitannins, gallotannins (EC(50) 8.5 to > 25 microg/ml) < C-glucosidic ellagitannins, dehydroellagitannins (EC(50) 0.6 - 2.8 microg/ml) at the host cell subtoxic concentration of 50 microg/ml. Furthermore, promastigotes of Leishmania donovani were assayed in the presence of these polyphenols and the results showed that none of the compounds was significantly toxic (EC(50) > 25 microg/ml) to the extracellular forms. In contrast, all polyphenols showed pronounced antileishmanial activities (EC(50) < 0.4 - 12.5 versus 7.9 microg/ml for Pentostam) against intracellular amastigotes of L. donovani residing within RAW cells. Noteworthy, most compounds exhibited low cytotoxicity against the murine host cells (EC(50) >25 microg/ml). Furthermore, some ellagitannins and the majority of dehydroellagitannins induced potent interferon-like activities as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on fibroblast L929 cells. This is the first report on hydrolyzable tannins as a new class of natural products with leishmanicidal activity including their potential for inducing the release of NO, TNF and IFN-like activity in macrophage-like RAW cells.     

Leishmanicidal activity of aurones

This is the first report on aurones as a new class of natural products with leishmanicidal activity. A series of aurones with drug-potential for Leishmania infections was identified in vitro using both a direct cytotoxicity test against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major, and a test against intracellular amastigote L. donovani residing within murine macrophages. The compounds proved to be active at concentrations in the microgram range between 0.4 and 5.0 microg/ml. When tested against murine bone marrow-derived macrophages as a mammalian host cell control, all compounds showed only moderate cytotoxicity (EC50 2.32-25.0 microg/ml).     

Antileishmanial and antibacterial activity of a new pyrazole derivative designated 4-[2-(1-(ethylamino)-2-methyl- propyl)phenyl]-3-(4-methyphenyl)-1-phenylpyrazole

Here, we report for the first time the synthesis and the antileishmanial activity of a new pyrazole derivative, namely 4-[2-(1-(ethylamino)-2-methylpropyl)phenyl]-3-(4-methyphenyl)-1-phenylpyrazole). Micromolar concentrations of this compound were found to inhibit the in vitro multiplication of Leishmania tropica, Leishmania major, and Leishmania infantum, three species causing different forms of leishmaniasis. Furthermore, the 50% inhibitory concentration (IC50) values for the compound are only slightly higher than those of amphotericin B, one of the most active antileishmanial agents used as a satisfactory substitute in cases not responding to pentostam. The IC50 values after 48 h for L. tropica, L. major, and L. infantum promastigote growth were 0.48 microg/mL, 0.63 microg/mL and 0.40 microg/mL, respectively for the compound, while they were 0.23 microg/mL, 0.29 microg/mL and 0.24 microg/mL, respectively for amphotericin B. We also tested this compound for its antibacterial activity against several bacteria. The strongest antibacterial activity was observed against Entrococcus feacalis and Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 60 microg/mL.     

In vitro action of platinum (II) and platinum (IV) complexes on Trypanosoma cruzi and Leishmania donovani

The in vitro activities of 22 neutral Pt(II) complexes, 4 Pt(IV) complex salts, and one Pt(II) complex salt on epimastigote forms of Trypanosoma cruzi and promastigote forms of Leishmania donovani were studied. Only 2 out of the 18 complexes with cis-Pt(DDH)Xn structure completely inhibited the growth of the epimastigote forms of T. cruzi and the promastigote of L. donovani. Against T. cruzi the cis-Pt(L)n(Cl)2 complexes showed no activities, but the complex in which the (L) ligand was stilbamidine did show a limited activity against L. donovani. Of the Pt(IV)complex salts, [Pt X6]H2(L) were very active against the epimastigote forms of T. cruzi. However, the Pt(II) complex salt [cis-Pt Cl4]H2 (pentamidine) showed no activity. The actions of the Pt(IV) complex salts on the promastigote forms of L. donovani were different and only those salts in which the (L) ligand was pentamidine or stilbamidine induced parasite growth inhibition. The one Pt(II) salt tested showed no antiparasitic activity. At the same time, the known antiparasitic activities of the nifurtimox and pentamidine molecules were confirmed.     

In vitro leishmanicidal activity of aurones.

A series of aurones with drug-potential for Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major, and a test against intracellular amastigote forms of L. donovani residing within murine macrophages. The most active aurone (6-hydroxy-2-[phenylmethylene]-3(2H)-benzofuranone) had an EC50 of 0.45 microgram/ml in the extra-, and an EC50 of 1.40 micrograms/ml in the intracellular assay. Other aurones were active between 0.06-12.50 micrograms/ml and 0.04-7.81 micrograms/ml, respectively. When tested against murine bone marrow-derived macrophages as a mammalian host cell control, the compounds showed only moderate cytotoxicity (EC50 2.32 to > 25.0 micrograms/ml). This is the first report on aurones as a new class of natural products with leishmanicidal activity.     

Toxicity of betulin derivatives and in vitro effect on promastigotes and amastigotes of Leishmania infantum and L. donovani

The toxicity and antileishmanial activity of 20 betulin derivatives were studied. The toxicity of betulin and synthesized compounds was determined using a bacterial test (Microtox) and two mammalian cell lines (CHO-K1 and J774). The antileishmanial activity of compounds (50 μM) was examined in both the promastigote and intracellular amastigote stages of Leishmania infantum and L. donovani. No correlation was found among the toxicity tests. All the compounds showed significant antipromastigote activity. The antiproliferative capacity of derivatives was dependent on the parasite stage studied, and no substantial differences were found between Leishmania species. Betulin, 3,28-di-O-acetylbetulin and L-aspartyl amide of betulonic acid showed moderate activity against amastigotes. The highest inhibition of intracellular amastigote multiplication was achieved with a low micromolar concentration (IC(50) ca 9 μM) of heterocyclic betulin derivative 3,28-di-O-acetyllup-13(18)-ene with N-ethyltriazolo moiety 16, without significant toxicity for mammalian cells. These results point to the interest of this lead compound for further in vitro and in vivo tests.     

Activities of Pt(II) and Ru(III) triazole-pyrimidine complexes against Trypanosoma cruzi and T. brucei brucei

We studied the biological activity of three newly synthesized metal complexes of triazole-pyrimidine derivatives that were previously observed to inhibit in vitro growth of epimastigotes of Trypanosoma cruzi and procyclic forms of Trypanosoma brucei brucei. We analyzed the possible inhibitory effect of these compounds on the synthesis of DNA, RNA and protein, ultrastructure and excretion of metabolites by these parasites. RNA synthesis was inhibited by all three complexes assayed. These complexes also led to anomalies of the main organelles (e.g. nucleus, kinetoplast and mitochondria). In addition, these complexes may be capable of altering the excretion of metabolites by the parasites.     

In vitro activity of C20-diterpenoid alkaloid derivatives in promastigotes and intracellular amastigotes of Leishmania infantum

The in vitro anti-proliferative effects are described of several atisine-type diterpenoid alkaloids against the protozoan parasite Leishmania infantum, which causes human visceral leishmaniasis and canine leishmaniasis in the Mediterranean basin, as well as human cutaneous leishmaniasis throughout the Mediterranean region. From a total of 43 compounds tested, including C19- and C20-diterpene alkaloids from several chemical classes, only 15,22-O-diacetyl-19-oxo-dihydroatisine, azitine and isoazitine were highly active against cultures of the parasite (promastigote form) with IC50 values within the range of the reference drug pentamidine-isothionate (7.39-12.80 mg/L for the test compounds, 11.32 mg/L for the positive control). These compounds were not toxic to the host cell. When treated with a dosage of 5 microg/mL of the active compounds (half of their IC50), the promastigote forms lost 80% of their infection capacity and the multiplication of extracellular forms of L. infantum was severely affected. The study showed that atisine-type C20-diterpenoid alkaloids exhibited promising anti-leishmanial properties with strong molecular selectivity. These might have implications for other intracellular pathogens- or phylogenetically related parasites, such as Trypanosoma spp.     

Synthesis and evaluation of antiparasitic activities of new 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives

A series of new 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives, prepared by two synthetic routes, were in vitro assayed against three Trypanosoma strains, Leishmania donovani, and Plasmodium falciparum K1. Seven out of 17 compounds showed moderate to very good activity against blood stage T. b. rhodesiense, with 10 and 17 exhibiting highest potency (IC50 of 1.0 and 1.1 microM, respectively). Interestingly, the beta-diketone precursors 1-3 had good antitrypanosomal activity toward the insect stage, with IC50 values of 1.0-3.4 microM. Among different compounds with moderate activity against T. cruzi, compound 17 showed the lowest IC50 value of 9.5 microM; thus, the series seemed to act selectively toward the different Trypanosoma parasites. Eight compounds were moderately active against L. donovani, with 2, 3, and 12 being the most promising ones (IC50 values of 2.3-5.2 microM), whereas compound 14 was the only derivative with good activity against P. falciparum (IC50 of 3.7 microM).     

Preliminary screening of antiprotozoal activity of Jasonia glutinosa aerial parts

As a part of a research on anti-protozoan agents, the acetonic extracts from Jasonia glutinosa aerial parts (Asteraceae) were tested in vitro against Entamoeba histolytica, Leishmania donovani (promastigote forms) and Trichomonas vaginalis. The 20% acetone extract was not effective at the assayed concentrations. The pure acetone extract was found to be active against Leishmania donovani and Entamoeba histolytica.     

In vitro activities of thiadiazine derivatives against Leishmania amazonensis

Ten thiadiazine derivatives were tested in vitro for antiparasitic effects against both extracellular promastigotes and intracellular amastigotes of Leishmania amazonensis. The results showed that the evaluated compounds exhibited a strong antiproliferative activity on all developmental stages of the parasite. The minimal inhibitory concentration and the 50 % effective concentration values against the promastigote were 2.1-5.1 microg/ml and 0.6-1.8 microg/ml, respectively. The tested compounds caused an irreversible inhibition of the promastigote growth either after 1 h of treatment with 10 microg/ml or after 24 h with 1 microg/ml. Also, the thiadiazine derivatives were active against amastigotes producing between 12 and 89 % of reduction of infection at 100 ng/ml. However, the compounds exhibited high toxicity and provoked inhibition of the phagocytosis in the murine host cell.     

Antiprotozoal activity of sesquiterpenes from jasonia glutinosa

Two sesquiterpene alcohols, [11 R]-eudesm-4(14)-en-5?,11,12-triol ( 1 ) and [11R]-eudesm-4(14)-en-5a,11-12-triol ( 2 ), from Jasonia glutinosa aerial parts (Asteraceae), have been tested using an in vitro technique against Leishmania donovani (promastigote forms) and Plasmodium falciparum . Sesquiterpene 1 was not effective at the assayed concentrations. Sesquiterpene 2 was found to be active against Leishmania donovani and Plasmodium falciparum .     

Antiprotozoal Activity of Sesquiterpenes from Jasonia Glutinosa

Two sesquiterpene alcohols, [11 R]-eudesm-4(14)-en-5ß,11,12-triol ( 1 ) and [11R]-eudesm-4(14)-en-5a,11-12-triol ( 2 ), from Jasonia glutinosa aerial parts (Asteraceae), have been tested using an in vitro technique against Leishmania donovani (promastigote forms) and Plasmodium falciparum . Sesquiterpene 1 was not effective at the assayed concentrations. Sesquiterpene 2 was found to be active against Leishmania donovani and Plasmodium falciparum .     

In vitro effect of new formulations of amphotericin B on amastigote and promastigote forms of Leishmania infantum

The in vitro antileishmanial activities of various new amphotericin B (AMB) formulations were investigated, including microspheres of hydrophilic albumin with three AMB aggregation forms (monomeric, dimeric and multiaggregate) and the polymers of polylactic-co-glycolic acid, Resomer RG502 and RG503 with the multiaggregate AMB form. This in vitro study was performed on the extracellular promastigote form and the intracellular amastigote form of a canine strain of Leishmania infantum (UCM 20) using the infected J774 murine macrophage-like cell line. Albumin-encapsulated forms did not show any toxicity for murine cells and had lower median effective concentration (EC50) values (ca. 0.003 microg/mL) for L. infantum amastigotes than free formulations (0.03 microg/mL). In addition, the aggregation state of AMB had a notable effect on the antileishmanial activity of the drug. Results obtained in vitro point towards interest in monomeric AMB encapsulated in microspheres in the chemotherapeutic control of leishmaniasis.     

Design,synthesis, and in vitro biological evaluation of novel thiazolopyrimidine derivatives as antileishmanial compounds

A series of thiazolopyrimidine derivatives was designed and synthesized as a Leishmania major pteridine reductase 1 (LmPTR1) enzyme inhibitor. Their LmPTR1 inhibitor activities were evaluated using the enzyme produced by Escherichia coli in a recombinant way. The antileishmanial activity of the selected compounds was tested in vitro against Leishmania sp. Additionally, the compounds were evaluated for cytotoxic activity against the murine macrophage cell line RAW 264.7. According to the results, four compounds displayed not only a potent in vitro antileishmanial activity against promastigote forms but also low cytotoxicity. Among them, compound L16 exhibited an antileishmanial activity for both the promastigote and amastigote forms of L. tropica, with IC₅₀ values of 7.5 and 2.69 µM, respectively. In addition, molecular docking studies and molecular dynamics simulations were also carried out in this study. In light of these findings, the compounds provide a new potential scaffold for antileishmanial drug discovery.     

Synergism in vitro of lovastatin and miconazole as anti-leishmanial agents.

The antifungal drug miconazole and the cholesterol-lowering agent lovastatin (mevinolin) were used in combination to assess their potency as anti-leishmanial agents. The drug combination was synergistic, being more potent in terms of inhibition of promastigote proliferation, macrophage infection and amastigote numbers. In promastigote cultures the effect was more marked in Leishmania amazonensis than L. donovani. Analysis of the sterol compositions of both promastigote and amastigote cultures revealed the inhibition of sterol 14 alpha-demethylation by miconazole and showed some apparent evidence of inhibition of sterol biosynthesis by lovastatin.     

In-vitro antileishmanial and trypanocidal activities of arsonoliposomes and preliminary in-vivo distribution in BALB/c mice

We have studied the antiprotozoal activity of some recently prepared and characterized arsonoliposome formulations. Plain arsonoliposomes and phosphatidylcholine arsonoliposomes prepared with palmitoyl- (C16) or lauroyl-(C12) acyl side chain arsonolipids showed in-vitro antileishmanial activity after a 72-h incubation period against wild-type promastigote forms of Leishmania donovani. The IC50 values ranged from 0.40 to 11.6 microM arsonolipid. Interestingly, all preparations tested were found to be significantly more potent against amphotericin B- or miltefosine-resistant promastigote forms of L. donovani, with IC50 values ranging between 0.21- and 2.33 microM arsonolipid. When tested in-vitro against Trypanosoma brucei brucei, all arsonoliposome formulations were found to have anti-trypanosomal activity after a 24-h incubation period. The fact that the corresponding arsonolipids (dissolved in dimethyl sulfoxide) were found not to be potent against the Leishmania promastigotes or the trypanosomes tested suggested that the formation of liposomes possibly influenced the mode of interaction between the active lipid and the parasites modulating their potency. In addition, a preliminary in-vivo study in BALB/c mice was performed for the initial evaluation of the biodistribution of arsonoliposomes. The accumulation of arsenic in the BALB/c mouse liver in relatively high amounts was an additional advantage of this approach for anti-protozoal therapy, especially for visceral leishmaniasis where parasites are located mainly in the liver.     

Design,synthesis, antileishmanial,and antifungal biological evaluation of novel 3,5-disubstituted isoxazole compounds based on 5-nitrofuran scaffolds

Nineteen 3,5-disubstituted-isoxazole analogs were synthesized based on nitrofuran scaffolds, by a [3 + 2] cycloaddition reaction between terminal acetylenes and 5-nitrofuran chloro-oxime. The compounds were obtained in moderate to very good yields (45–91%). The antileishmanial activity was assayed against the promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. Alkylchlorinated compounds 14p–r were active on both the promastigote and amastigote forms, with emphasis on compound 14p , which showed strong activity against the amastigote form (IC₅₀ = 0.6 μM and selectivity index [SI] = 5.2). In the alkyl series, compound 14o stands out with an IC₅₀ = 8.5 μM and SI = 8.0 on the amastigote form. In the aromatic series, the most active compounds were those containing electron-donor groups, such as trimethoxy isoxazole 14g (IC₅₀ = 1.2 μM and SI = 20.2); compound 14h , with IC₅₀ = 7.0 μM and SI = 6.1; and compound 14j containing the 4-SCH₃ group, with IC₅₀ = 5.7 μM and SI = 10.2. In addition, the antifungal activity of 19 nitrofuran isoxazoles was evaluated against five strains of Candida (C. albicans, C. parapsilosis, C. krusei, C. tropicalis, and C. glabrata). Eleven isoxazole derivatives were active against C. parapsilosis, and compound 14o was found to be the most active (minimal inhibitory concentration [MIC] = 3.4 μM) for this strain. Compound 14p was active against all the strains tested, with an MIC = 17.5 μM for C. glabrata, lower than that of the fluconazole used as the reference drug.     

In vitro and in vivo activities of three acridine thioethers against Leishmania donovani

The lack of definitive chemotherapeutic agents to fight against visceral leishmaniasis has lead to the testing of numerous compounds. In the present work, we carry out an in-depth study of the activity against Leishmania donovani of three acridine derivatives both in vitro and in vivo. These compounds have proven to be highly effective at medium and high concentrations of 10 microg/ml, against both flagellate and nonflagellate forms of the parasite, which, though obtained in vitro, closely resemble natural intracellular amastigotes. The in vivo assays showed a significant reduction in the percentage of parasitation versus control, for all the compounds tested. In addition, we have studied the possible mechanism by which these acridine derivatives act: they displayed a greater inhibitory effect against macromolecule synthesis in treated flagellates, yet alterations are also caused in the production of end metabolites and in the activity of different enzymes. The data obtained indicate that the acridine derivatives had several targets, one of them is the synthesis of nucleic acids and proteins, while the second one might be interaction with the carbohydrate and energy-production processes in the parasite. This conclusion is consistent with our observations concerning the ultrastructural changes induced in the parasite by these compounds principally at the mitochondrial level.     

Cytotoxicity of (2,2':6',2'-terpyridine)platinum(II) complexes to Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei

A range of (2,2':6',2'-terpyridine)platinum(II) complexes are shown to possess antiprotozoal activity in vitro against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei,the causative organisms of tropical diseases leishmaniasis and trypanosomiasis. The best compounds caused 100% and 78% inhibition of growth of the intracellular amastigote forms of L. donovani and T. cruzi, respectively, at a concentration of 1 microM and 100% inhibition of growth of the bloodstream trypomastigote forms of T. brucei at a concentration of 0.03 microM. The results obtained with complexes in which the fourth ligand to platinum(II) is capable of being substituted with a substitution inert hydroxyethanethiolate complex are compared. The ammine complexes show high antiprotozoal activity suggesting that the trans influence of the 2,2':6',2'-terpyridine ligand has a profound effect on the ease of displacement of the fourth ligand in (2,2':6',2' -terpyridine)platinum(II) complexes, although nonbonded interaction between the ammine ligand and the 6 and 6' ' hydrogens probably also weakens the ligation to Pt(II).