Synthesis and preliminary evaluation of new ursolic and oleanolic acids derivatives as antileishmanial agents

A series of new ursolic and oleanolic acids derivatives was synthesized via ursolic or oleanolic acids, previously extracted from South American Ilex species. These new compounds were tested for in vitro antiparasitic activity on Leishmania amazonensis and Leishmania infantum strains. Some of these compounds showed activity against the promastigote forms of L. amazonensis or L. infantum, with IC(50) ranging from 5 to 12 microM. As expected, most of the compounds showed a significant level of cytotoxicity against monocytes (IC(50) = 2-50 microM). From a structure-activity relationships point of view, these pharmacological results enlightened mainly the importance of an acetylation at position 3 of the oleanolic acid skeleton in the activity against the L. amazonensis strain, and of a bis-(3-aminopropyl)piperazine moiety on the carboxylic function of ursolic acid against the L. infantum strain.     

Synthesis of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxalines as antileishmanial agents by Suzuki-Miyaura cross-coupling reactions

A series of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives, structural analogues of alkaloid chimanine B, was synthesized in good yields using efficient palladium(0)-catalyzed Suzuki-Miyaura cross-coupling reactions. These new compounds were tested for in vitro antiparasitic activity upon Leishmania amazonensis and Leishmania infantum strains. Biological results showed activity against the promastigote forms of L. amazonensis and L. infantum with IC50 ranging from 0.5 to 7 microM. From a Structure-Activity Relationships point of view, these pharmacological results mainly enlightened the importance of the 4-lateral C6, C7 or C8 alpha-unsaturated trans-alkenyl chain of unsubstituted pyrrolo[1,2-a]quinoxaline moiety.     

Antileishmanial activity of lapachol analogues

The antileishmanial activity of lapachol, isolapachol, and dihydrolapachol, along with soluble derivatives (potassium salt) and acetate was obtained. All the compounds were assayed against metacyclic promastigotes of two different species of Leishmania associated to tegumentar leishmaniasis: L. amazonensis and L. braziliensis. All compounds presented significant activity, being isolapachol acetate the most active against promastigotes, with IC50/24h = 1.6 +/- 0.0 microg/ml and 3.4 +/- 0.5 microg/ml for, respectively, L. amazonensis and L. braziliensis. This compound was also assayed in vivo against L. amazonensis and showed to be active. Its toxicity in vitro was also established, and at concentration similar to the IC50, no toxicity was evidenced. In all experiments, pentamidine isethionate was used as a reference drug. The present results reinforce the potential use of substituted hydroxyquinones and derivatives as promising antileishmanial drugs and suggest a continuing study within this class of compounds.     

Synthesis of steroidal quinones and hydroquinones from bile acids by Barton radical decarboxylation and benzoquinone addition. Studies on their cytotoxic and antifungal activities

Twelve new hydroquinones and quinones (4a-c to 7a-c) derived from free or peracetylated bile acids were prepared by a Barton decarboxylation reaction, with subsequent trapping of the resulting free radical by benzoquinone. All new compounds were completely characterized by 2D NMR techniques and screened for antifungal and cytotoxic activity. One of the new hydroquinones (7b) showed promising results against the human pancreatic ductal carcinoma cell line PANC1, with similar cytotoxic activity as the commercial chemotherapy drug doxorubicin.     

Antileishmanial activity of indole alkaloids from Aspidosperma ramiflorum

The present study was designated to evaluate the antileishmanial activity of acid and basic fractions that were obtained after acid-basic extraction, from ethanolic 70% crude extract and pure compounds from the stem bark of Aspidosperma ramiflorum. The basic alkaloidal fraction presented a good activity against the extracellular form (promastigotes) of Leishmania (L.) amazonensis (LD(50) value<47 microg/ml). Based on these findings, the basic fraction was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording individual purified ramiflorines A and B. Both ramiflorines A and B showed significant activity against Leishmania (L.) amazonensis (LD(50) values of 16.3+/-1.6 microg/ml and 4.9+/-0.9 microg/ml, respectively). Our results are promising, showing that these compounds are biologically active against Gram-positive bacteria.     

Effect of thiadiazine derivatives on intracellular amastigotes of Leishmania amazonensis

Current therapy for leishmaniasis is not satisfactory. We describe the in vitro antiproliferative effects of new thiadiazine derivatives against Leishmania amazonensis. The compounds were found to be active against the amastigote form of the parasite, inhibiting parasite growing, from 10 to 89%, at a concentration of 100 ng/ml. This activity suggests that thiadiazine derivatives could be considered as potential antileishmanial compounds.     

Trypanothione reductase activity is prominent in metacyclic promastigotes and axenic amastigotes of Leishmania amazonesis. Evaluation of its potential as a therapeutic target

The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N'-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment of leishmaniasis.     

Design, synthesis, and biological evaluation of hydroquinone derivatives as novel inhibitors of the sarco/endoplasmic reticulum calcium ATPase

Analogues of the compound 2,5-di-tert-butylhydroquinone (BHQ) are capable of inhibiting the enzyme sarco/endoplasmic reticulum ATPase (SERCA) in the low micromolar and submicromolar concentration ranges. Not only are SERCA inhibitors valuable research tools, but they also have potential medicinal value as agents against prostate cancer. This study describes the synthesis of 13 compounds representing several classes of BHQ analogues, such as hydroquinones with a single aromatic substituent, symmetrically and unsymmetrically disubstituted hydroquinones, and hydroquinones with ω-amino acid tethers attached to their hydroxyl groups. Structure–activity relationships were established by measuring the inhibitory potencies of all synthesized compounds in bioassays. The assays were complemented by computational ligand docking for an analysis of the relevant ligand/receptor interactions.     

Anti-leishmanial activity of alkaloidal extract from Aspidosperma ramiflorum

Infections due to protozoa of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials (SbV), which present renal and cardiac toxicity. Besides, the precise chemical structure and mechanism of action of these drugs are unknown up to date. In order to find new drugs against leishmaniasis, we have been studying extracts of Brazilian trees. In the present study, we have evaluated the effectiveness of an alkaloid extract of Aspidosperma ramiflorum Muell. Arg. (Apocynaceae), against the extracellular forms promastigotes of L. (L.) amazonensis and L. (V.) braziliensis. The alkaloid extract of A. ramiflorum was much more effective against L. (L.) amazonensis (LD50 < 47 microg/ml) than L. (V.) braziliensis. Based on these in vitro results against L. (L.) amazonensis new studies should be made to find the compounds with anti-leishmanial activity.     

Studies on the effectiveness of diarylheptanoids derivatives against Leishmania amazonensis

In a previous work we demonstrated that diarylheptanoids extracted from Centrolobium sclerophyllum are very active against Leishmania amazonensis promastigotes. In order to continue our studies with these class of compounds, we decided to evaluate the activity of several diarylheptanoids derived from curcumin (diferuloyl methane) against the extracellular form (promastigotes) of L. amazonensis. Furthermore, an experiment against the intracellular form of the parasite (amastigotes) was carried out, comparing the most active compound among the curcumin derivatives (the methylcurcumin) with des-O-methylcentrolobine, the most active diarylheptanoid derived from C. sclerophyllum.     

The efficacy of 2-nitrovinylfuran derivatives against Leishmania in vitro and in vivo

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC₅₀) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC₅₀ and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural^(r) (a furvina-containing antifungal ointment) for the treatment of CL.     

Studies on quinones. Part 42: Synthesis of furylquinone and hydroquinones with antiproliferative activity against human tumor cell lines

The preparation of furyl-1,4-quinone and hydroquinones by reaction of 2-furaldehyde N,N-dimethylhydrazone with benzo- and naphthoquinones is reported. Access to furylnaphthoquinones from unactivated quinones requires acid-induced conditions, however oxidative coupling reactions of activated quinones proceed under neutral conditions. The in vitro cytotoxic activity of the prepared compounds against a panel of three human cancer cell lines has been studied. Most of the furyl-1,4-quinones exhibited good antiproliferative activity (GI(50)=6.5-33.5microm) against the MCF-7, NCI-H460, and SF-268 (CNS cancer) cell lines chosen for testing.     

Therapeutic switching: from antidermatophytic essential oils to new leishmanicidal products

This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC₅₀) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC₅₀: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis.     

Leishmania amazonensis: Biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities

The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages.     

A new heptasubstituted (E)-aurone glucoside and other aromatic compounds of Gomphrena agrestis with biological activity

A new aurone 1 and two known substances, aurantiamide acetate (2) and tiliroside (3), were isolated from the ethanolic extract of Gomphrena agrestis. The structural determination of 1 was based on spectroscopic and spectrometric data. The substance was defined as (E)-3'-O-beta-D-glucopyranosyl-4,5,6,4'-tetrahydroxy-7,2'-dimethoxyaurone. Biological activity of the ethanolic crude extract and isolated compounds against bacteria, fungi and Leishmania amazonensis amastigotes was evaluated. This appears to be the first report documenting aurone and aurantiamide compounds in the Amaranthaceae family. In the evaluation of biological activity the ethanolic extract of G. agrestis and compounds 1, 2, and 3 were shown to be active mainly against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa.     

Design, synthesis and antileishmanial in vitro activity of new series of chalcones-like compounds: a molecular hybridization approach

The chalcone-like series 1a-1g was efficiently synthesized from Morita-Baylis-Hillman reaction (52-74% yields). Compounds 1a-1g were designed by molecular hybridization based on the anti-inflammatory drug methyl salicylate (3) and the antileishmanial moiety of the Morita-Baylis-Hillman adducts 2a-2g. The 1a-1g compounds were much more actives than precursor series 2a-2g, for example, IC(50)=7.65 μM on Leishmania amazonensis and 10.14 μM on Leishmania chagasi (compound 1c) when compared to IC(50)=50.08 μM on L. amazonensis and 82.29 μM on L. chagasi (compound 2c). The IC(50) values of compound 3 (228.49 μM on L. amazonensis and 261.45 μM on L. chagasi) and acryloyl salicylate 4 (108.50 μM on L. amazonensis and 118.83 μM on L. chagasi) were determined here, by the first time, on Leishmania.     

Anti-leishmanial activity of alkaloidal extracts obtained from different organs of Aspidosperma ramiflorum

The present study was designated to evaluate semi-quantitative antileishmanial activity of alkaloidal extracts that were obtained from 1g of different parts of Aspidosperma ramiflorum (leaves, roots, seeds, and stem barks). Alkaloidal extracts of barks and leaves presented a good activity against the extracellular form (promastigotes) of Leishmania (L.) amazonensis. It is known that compounds responsible for the antileishmanial activity in the alkaloidal extracts from A. ramiflorum are the monoterpenoid indole alkaloids ramiflorine A and ramiflorine B, therefore extracts obtained from different plant parts were analyzed by electrospray ionization mass spectrometry (ESI-MS) in order to evidence the presence of these bioactive alkaloids. Based on these findings, alkaloidal extract from leaves was fractionated on preparative thin-layer chromatography in a bioassay-guided fractionation affording individual purified ramiflorines A and B. Both ramiflorines A and B showed significant activity against Leishmania (L.) amazonensis (LD(50) values of 18.5±6.5μg/ml and 12.63±5.52μg/ml, respectively). Our results are showing that alkaloidal extract from leaves is a promising alternative to the use of stem barks from A. ramiflorum.     

Bioactivity of crude extracts and some constituents of Blutaparon portulacoides (Amaranthaceae).

Crude extracts (aerial parts and roots, both dried), methylenedioxyflavonol, and a mixture of acyl steryl glycosides isolated from Blutaparon portulacoides, were assayed for their toxicity against Trypanosoma cruzi trypomastigotes and Leishmania amazonensis amastigotes from axenic cultures. The antimicrobial activity was also investigated, in a screening conducted using fifteen strains of Gram-positive and Gram-negative bacteria, along with the yeasts, Candida albicans and Candida tropicalis. To assess the antibacterial activity of the isolated compounds, the minimum inhibitory concentrations (MICs) were determined. There are no reports of acyl steryl glycosides in the genus Blutaparon and their biological activities are being evaluated for the first time.     

Antioxidant activity of prenylated hydroquinone and benzoic acid derivatives from Piper crassinervium Kunth

Growing evidence suggests that RNOS (reactive nitrogen and oxygen species) are involved in the damage of biomolecules, contributing to the aetiology of several human diseases. Thus, the demand for antioxidants has stimulated the search for new compounds with potential use in this field. The in vitro antioxidant potential of prenylated hydroquinones and prenylated 4-hydroxy-benzoic acids from fruits of P. crassinervium was evaluated in terms of their capacity to suppress both DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and chemiluminescence produced from luminol, using 2,2'-azo-bis(2-amidinopropane) (ABAP) as a peroxyl radical source. The inhibition of lipid peroxidation was assessed using liposomes from phosphatidylcholine as a membrane model. The prenylated hydroquinones had higher antioxidant activity than the benzoic acids and, among the hydroquinones, the E isomer was more efficient than the Z isomer.     

Antileishmanial activity of diterpene acids in copaiba oil

Leishmaniasis is a neglected tropical disease. According to the World Health Organization, there are approximately 1.5-two million new cases of cutaneous leishmaniasis each year worldwide. Chemotherapy against leishmaniasis is based on pentavalent antimonials, which were developed more than a century ago. The goals of this study were to investigate the antileishmanial activity of diterpene acids in copaiba oil, as well as some possible targets of their action against Leishmania amazonensis. Methyl copalate and agathic, hydroxycopalic, kaurenoic, pinifolic and polyaltic acids isolated from Copaifera officinales oleoresins were utilised. Ultrastructural changes and the specific organelle targets of diterpenes were investigated with electron microscopy and flow cytometry, respectively. All compounds had some level of activity against L. amazonensis. Hydroxycopalic acid and methyl copalate demonstrated the most activity against promastigotes and had 50% inhibitory concentration (IC₅₀) values of 2.5 and 6.0 µg/mL, respectively. However, pinifolic and kaurenoic acid demonstrated the most activity against axenic amastigote and had IC₅₀ values of 3.5 and 4.0 µg/mL, respectively. Agathic, kaurenoic and pinifolic acid caused significant increases in plasma membrane permeability and mitochondrial membrane depolarisation of the protozoan. In conclusion, copaiba oil and its diterpene acids should be explored for the development of new antileishmanial drugs.