Antileishmanial Effects of Acetylene Acetogenins from Seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae) and Semisynthetic Derivatives.

As part of our continuous studies involving the prospection of natural products from Brazilian flora aiming at the discovery of prototypes for the development of new antiparasitic drugs, the present study describes the isolation of two natural acetylene acetogenins, (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11'-yn-19'-enyl)butanolide (1) and (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11'-ynyl)butanolide (2), from the seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae). Using an ex-vivo assay, compound 1 showed an IC50 value of 29.9 µM against the intracellular amastigote forms of Leishmania (L.) infantum, whereas compound 2 was inactive. These results suggested that the terminal double bond plays an important role in the activity. This effect was also observed for the semisynthetic acetylated (1a and 2a) and eliminated (1b and 2b) derivatives, since only compounds containing a double bond at C-19 displayed activity, resulting in IC50 values of 43.3 µM (1a) and 23.1 µM (1b). In order to evaluate the effect of the triple bond in the antileishmanial potential, the mixture of compounds 1 + 2 was subjected to catalytic hydrogenation to afford a compound 3 containing a saturated side chain. The antiparasitic assays performed with compound 3, acetylated (3a), and eliminated (3b) derivatives confirmed the lack of activity. Furthermore, an in-silico study using the SwissADME online platform was performed to bioactive compounds 1, 1a, and 1b in order to investigate their physicochemical parameters, pharmacokinetics, and drug-likeness. Despite the reduced effect against amastigote forms of the parasite to the purified compounds, different mixtures of compounds 1 + 2, 1a + 2a, and 1b + 2b were prepared and exhibited IC50 values ranging from 7.9 to 38.4 µM, with no toxicity for NCTC mammalian cells (CC50 > 200 µM). Selectivity indexes to these mixtures ranged from >5.2 to >25.3. The obtained results indicate that seeds of Porcelia macrocarpa are a promising source of interesting prototypes for further modifications aiming at the discovery of new antileishmanial drugs.

Antiproliferative and Genotoxic Action of an Underexploited Organoteluran Derivative on Sarcoma 180 Cells.

BACKGROUND: The search for novel metallic chemical compounds with toxicogenic effects has been of great importance for more efficient cancer treatment. OBJECTIVE: The study evaluated the cytotoxic, genotoxic and mutagenic activity of organoteluran RF07 in the S-180 cell line. METHODS: The bioassays used were cell viability with 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, evaluation of apoptosis and necrosis using fluorescence and flow cytometry, cytokinesisblock micronucleus test and comet assay. The compound was tested at 1; 2.5 and 5µM. RESULTS: The results showed the cytotoxicity of RF07 at concentrations of 2.5, 5, 10 and 20µM when compared to the negative control. For genotoxicity tests, RF07 showed effects in all concentrations assessed by increased index and frequencies of damage and mutagenic alterations. The compound was also cytotoxic due to the significant decrease in the nuclear division index, with significant values of apoptosis and necrosis. The results of fluorescence and flow cytometry showed apoptosis as the main type of cell death caused by RF07 at 5µM, which is thought to avoid an aggressive immune response of the organism. CONCLUSION: In addition to cytotoxic and genotoxic effects, RF07 creates good perspectives for future antitumor formulations.

preADMET analysis and clinical aspects of dogs treated with the Organotellurium compound RF07: A possible control for canine visceral leishmaniasis?

Tellurium compounds have been described as potential leishmanicides, bearing promising leishmanicidal and antimalarial effects. Therefore, the present study investigated the pharmacological potential of the organotellurane compound RF07 through preADMET parameters, such as absorption, distribution, metabolism and excretion. After studying the pharmacokinetic properties of RF07, studies were carried out on dogs naturally infected with visceral leishmaniasis after the administration of RF07, in order to assess pathophysiological parameters. Thus, dogs were divided into 4 groups with administration of daily intraperitoneal injections for 3 weeks (containing RF07 or placebo). During the trial, hematological parameters, renal and hepatic toxicity were evaluated. Serum urea, creatinine, alkaline phosphatase, transaminases (GOT and GPT), as well as hemogram results, were evaluated before the first administration and during the second and third weeks after the start of the treatment. In dogs with VL, RF07 improved liver damage, regulated GPT levels and significantly decreased leukocyte count, promoting its regularization. These phenomena occurred at the end of the third week of treatment. The administration of RF07 promoted a significant decrease in the average levels of GOT and GPT after the third week of treatment and did not significantly alter the hematological parameters. The application of RF07 in the treatment of visceral leishmaniasis suggests that it is an alternative to the disease, since the reversal of clinical signs in dogs with VL requires the use of 0.6 mg/kg.

CPP-Ala-Ala-Tyr-PABA inhibitor analogs with improved selectivity for neurolysin or thimet oligopeptidase.

Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (NEL, EC 3.4.24.16) are closely related zinc-dependent metalo-oligopeptidases, which take part in the metabolism of oligopeptides (from 5 to 17 amino acid residues) inside and outside cells. Both peptidases are ubiquitously distributed in tissues. TOP is one of the main intracellular peptide-processing enzymes being important for the antigen selection in the MHC Class I presentation route, while NEL function has been more associated with the extracellular degradation of neurotensin. Despite efforts being made to develop specific inhibitors for these peptidases, the most used are: CPP-Ala-Ala-Tyr-PABA, described by Orlowski et al. in 1988, and CPP-Ala-Aib-Tyr-PABA (JA-2) that is an analog more resistant to proteolysis, which development was made by Shrimpton et al. in 2000. In the present work, we describe other analogs of these compounds but, with better discriminatory capacity to inhibit specifically NEL or TOP. The modifications introduced in these new analogs were based on a key difference existent in the extended binding sites of NEL and TOP: the negatively charged Glu469 residue of TOP corresponds to the positively charged Arg470 residue of NEL. These residues are in position to interact with the residue at the P1' and/or P2' of their substrates (mimicked by the Ala-Ala/P1'-P2' residues of the CPP-Ala-Ala-Tyr-PABA). Therefore, exploring this single difference, the following compounds were synthesized: CPP-Asp-Ala-Tyr-PABA, CPP-Arg-Ala-Tyr-PABA, CPP-Ala-Asp-Tyr-PABA, CPP-Ala-Arg-Tyr-PABA. Confirming the predictions, the replacement of each non-charged residue of the internal portion Ala-Ala by a charged residue Asp or Arg resulted in compounds with higher selectivity for NEL or TOP, especially due to the electrostatic attraction or repulsion by the NEL Arg470 or TOP Glu469 residue. The CPP-Asp-Ala-Tyr-PABA and CPP-Ala-Asp-Tyr-PABA presented higher affinities for NEL, and, the CFP-Ala-Arg-Tyr-PABA showed higher affinity for TOP.

Development of a methodology for reversible chemical modification of silicon surfaces with application in nanomechanical biosensors.

Hypervalent tellurium compounds have a particular reactivity towards thiol compounds which are related to their biological properties. In this work, this property was assembled to tellurium-functionalized surfaces. These compounds were used as linkers in the immobilization process of thiolated biomolecules (such as DNA) on microcantilever surfaces. The telluride derivatives acted as reversible binding agents due to their redox properties, providing the regeneration of microcantilever surfaces and allowing their reuse for further biomolecules immobilizations, recycling the functional surface. Initially, we started from the synthesis of 4-((3-((4-methoxyphenyl) tellanyl) phenyl) amino)-4-oxobutanoic acid, a new compound, which was immobilized on a silicon surface. In nanomechanical systems, the detection involved a hybridization study of thiolated DNA sequences. Fluorescence microscopy technique was used to confirm the immobilization and removal of the telluride-DNA system and provided revealing results about the potentiality of applying redox properties to chalcogen derivatives at surfaces.

Antitumor effect of chiral organotelluranes elicited in a murine melanoma model.

Protease roles in cancer progression have been demonstrated and their inhibitors display antitumor effects. Cathepsins are lysosomal cysteine proteases that have increased expression in tumor cells, and tellurium compounds were described as potent cysteine protease inhibitors and also assayed in several animal models. In this work, the two enantiomeric forms of 1-[Butyl(dichloro)-λ4-tellanyl]-2-[1S-methoxyethyl]benzene (organotelluranes RF-13R and RF-13S) were evaluated as inhibitors of cathepsins B and L, showing significant enantiodiscrimination. We observed their cytotoxic effects on a murine melanoma model, effectively inhibiting tumor progression in vivo. The enantiomers were able to inhibit melanoma cell viability, migration and invasion in vitro. Besides, RF-13S and RF-13R were able to inhibit endothelial cell angiogenesis using a tube formation assay in vitro, in a stereodependent manner. These organotelluranes affected cell morphology, showing disassembling of the actin cytoskeleton. These results suggest organotelluranes as potential antitumor agents, acting directly on tumor cell proliferation, migration and invasion, and on endothelial cells, disrupting angiogenesis, showing low toxicity and high efficiency. Taken together our results suggest that this class of compounds should be further studied to reveal their potential as antitumoral agents.

Molecular, Biological and Structural Features of VL CDR-1 Rb44 Peptide, Which Targets the Microtubule Network in Melanoma Cells.

Microtubules are important drug targets in tumor cells, owing to their role in supporting and determining the cell shape, organelle movement and cell division. The complementarity-determining regions (CDRs) of immunoglobulins have been reported to be a source of anti-tumor peptide sequences, independently of the original antibody specificity for a given antigen. We found that, the anti-Lewis B mAb light-chain CDR1 synthetic peptide Rb44, interacted with microtubules and induced depolymerization, with subsequent degradation of actin filaments, leading to depolarization of mitochondrial membrane-potential, increase of ROS, cell cycle arrest at G2/M, cleavage of caspase-9, caspase-3 and PARP, upregulation of Bax and downregulation of Bcl-2, altogether resulting in intrinsic apoptosis of melanoma cells. The in vitro inhibition of angiogenesis was also an Rb44 effect. Peritumoral injection of Rb44L1 delayed growth of subcutaneously grafted melanoma cells in a syngeneic mouse model. L1-CDRs from immunoglobulins and their interactions with tubulin-dimers were explored to interpret effects on microtubule stability. The opening motion of tubulin monomers allowed for efficient L1-CDR docking, impairment of dimer formation and microtubule dissociation. We conclude that Rb44 VL-CDR1 is a novel peptide that acts on melanoma microtubule network causing cell apoptosis in vitro and melanoma growth inhibition in vivo.

Blockade of MIF-CD74 Signalling on Macrophages and Dendritic Cells Restores the Antitumour Immune Response Against Metastatic Melanoma.

Mounting an effective immune response against cancer requires the activation of innate and adaptive immune cells. Metastatic melanoma is the most aggressive form of skin cancer. While immunotherapies have shown a remarkable success in melanoma treatment, patients develop resistance by mechanisms that include the establishment of an immune suppressive tumor microenvironment. Thus, understanding how metastatic melanoma cells suppress the immune system is vital to develop effective immunotherapies against this disease. In this study, we find that macrophages (MOs) and dendritic cells (DCs) are suppressed in metastatic melanoma and that the Ig-CDR-based peptide C36L1 is able to restore MOs and DCs' antitumorigenic and immunogenic functions and to inhibit metastatic growth in lungs. Specifically, C36L1 treatment is able to repolarize M2-like immunosuppressive MOs into M1-like antitumorigenic MOs, and increase the number of immunogenic DCs, and activated cytotoxic T cells, while reducing the number of regulatory T cells and monocytic myeloid-derived suppressor cells in metastatic lungs. Mechanistically, we find that C36L1 directly binds to the MIF receptor CD74 which is expressed on MOs and DCs, disturbing CD74 structural dynamics and inhibiting MIF signaling on these cells. Interfering with MIF-CD74 signaling on MOs and DCs leads to a decrease in the expression of immunosuppressive factors from MOs and an increase in the capacity of DCs to activate cytotoxic T cells. Our findings suggest that interfering with MIF-CD74 immunosuppressive signaling in MOs and DCs, using peptide-based immunotherapy can restore the antitumor immune response in metastatic melanoma. Our study provides the rationale for further development of peptide-based therapies to restore the antitumor immune response in metastatic melanoma.

Antitrypanosomal activity and evaluation of the mechanism of action of dehydrodieugenol isolated from Nectandra leucantha (Lauraceae) and its methylated derivative against Trypanosoma cruzi.

BACKGROUND: From a previous screening of Brazilian biodiversity for antiprotozoal activity, the hexane extract from leaves of Nectandra leucantha (Nees & Mart.) (Lauraceae) demonstrated activity against Trypanosoma cruzi. Chromatographic separation of this extract afforded bioactive dehydrodieugenol (1). Furthermore, methylated derivative 2 (dehydrodieugenol dimethyl ether) was prepared and also tested against T. cruzi. PURPOSE: To examine the therapeutical potential of compounds 1 and 2 against T. cruzi as well as to elucidate the mechanism of action of bioactive compound 1 against T. cruzi. METHODS/STUDY DESIGN: Crude hexane extract from leaves was subjected to chromatographic steps to afford bioactive compound 1. In order to analyze the effect of additional methyl group in the antiparasitic activity of 1, derivative 2 was prepared (both are no pan-assay interference compounds - PAINS). These compounds were evaluated in vitro against T. cruzi (trypomastigote and amastigote forms) and analyzed for the potential effect in host cells through the production of nitric oxide and reactive oxygen species. Finally, the plasma membrane effect of the most potent compound 1 was investigated in T. cruzi trypomastigotes. RESULTS: Compounds 1 and 2 displayed activity against amastigotes of T. cruzi. Although both compounds promoted activity against intracellular amastigotes, the production of nitric oxide and reactive oxygen species of host cells were unaltered, suggesting an antiparasitic activity other than host cell activation. Considering 1 the most effective compound against T. cruzi, the interference in the plasma membrane of the trypomastigotes was investigated using the fluorescent probe SYTOX® Green. After a short-term incubation, the fluidity and integrity of the plasma membrane was completely altered, suggesting it as a primary target for compound 1 in T. cruzi. CONCLUSION: Compounds 1 and 2 selectively eliminated the intracellular parasites without host cell activation and could be important scaffolds for the search of new hit compounds.

Stability Study of Hypervalent Tellurium Compounds in Aqueous Solutions.

Hypervalent tellurium compounds (telluranes) are promising therapeutical agents with negligible toxicities for some diseases in animal models. The C-Te bond of organotellurium compounds is commonly considered unstable, disfavoring their applicability in biological studies. In this study, the stability of a set of telluranes composed of an inorganic derivative and noncharged and charged organic derivatives was monitored in aqueous media with 1H, 13C, and 125Te NMR spectroscopy and high-resolution mass spectrometry. Organic telluranes were found to be remarkably resistant and stable to hydrolysis, whereas the inorganic tellurane AS101 is totally converted to the hydrolysis product, trichlorooxytellurate, [TeOCl 3 ]-, which was also observed in the hydrolysis of TeCl 4 . The noteworthy stability of organotelluranes in aqueous media makes them prone to further structure-activity relationship studies and to be considered for broad biological investigations.

The Ig VH complementarity-determining region 3-containing Rb9 peptide, inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor.

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the VHCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca2+ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.

Hypervalent organotellurium compounds as inhibitors of P. falciparum calcium-dependent cysteine proteases.

Hypervalent organotellurium compounds (organotelluranes) have shown several promising applications, including their use as potent and selective cysteine protease inhibitors and antiprotozoal agents. Here, we report the antimalarial activities of three organotellurane derivatives (RF05, RF07 and RF19) in two Plasmodium falciparum strains (CQS 3D7 and CQR W2), which demonstrated significant decreases in parasitemia in vitro. The inhibition of intracellular P. falciparum proteases by RF05, RF07 and RF19 was determined and the IC50 values were 3.7±1.0µM, 1.1±0.2µM and 0.2±0.01µM, respectively. Using an assay performed in the presence of the ER Ca(2+)-ATPase inhibitor we showed that the main enzymatic targets were cysteine proteases stimulated by calcium (calpains). None of the compounds tested caused haemolysis or a significant decrease in endothelial cell viability in the concentration range used for the inhibition assay. Taken together, the results suggest promising compounds for the development of antimalarial drugs.

Effects of trichlorotelluro-dypnones on mitochondrial bioenergetics and their relationship to the reactivity with protein thiols.

The effect of four trichlorotelluro-dypnones, named compounds 1, 2, 3, and 4, on the bioenergetics of isolated rat liver mitochondria (RLM) and cells was investigated. In a dose-dependent manner, the studied organotelluranes promoted Ca(2+)-dependent mitochondrial swelling inhibited by cyclosporine A and were associated with a decrease of the total mitochondrial protein thiol content. These effects characterize the opening of the classical mitochondrial permeability transition pore. Despite the reactivity with mitochondrial protein thiol groups, these compounds did not promote significant glutathione depletion. In the absence of Ca(2+), the organotelluranes promoted mitochondrial loss of ΔΨ in RLM concomitant with respiratory control decrease due to an increase of the state 4 respiration rate. In these conditions, mitochondrial swelling was absent, and thiol content was higher than that in the presence of Ca(2+). The differentiated effects observed in the presence and absence of Ca(2+) are probably related to the effects of that ion on membrane structure, with repercussions for the exposure of specific reactive protein thiol groups. In smooth muscle cells, these compounds promoted the loss of mitochondrial ΔΨ and apoptosis. The loss of ΔΨ was not preceded by a decrease of cell viability that is consistent with mitochondria as the primary targets for the action of these organotelluranes.

Cytotoxicity of phenothiazine derivatives associated with mitochondrial dysfunction: a structure-activity investigation.

Phenothiazine derivatives are neuroleptic drugs used in the treatment of schizophrenia and anxiety. Several side effects are described for these drugs, including hepatotoxicity, which may be related to their cytotoxic activity. Working with isolated rat liver mitochondria, we previously showed that phenothiazine derivatives induced the mitochondrial permeability transition associated with cytochrome c release. Since the mitochondrial permeabilization process plays a central role in cell death, the aim of this work was to evaluate the effects of five phenothiazine derivatives (chlorpromazine, fluphenazine, thioridazine, trifluoperazine, and triflupromazine) on the viability of hepatoma tissue culture (HTC) cells to establish the structural requirements for cytotoxicity. All phenothiazine derivatives decreased the viability of the HTC cells in a concentration-dependent manner and exhibited different cytotoxic potencies. The EC50 values ranged from 45 to 125 µM, with the piperidinic derivative thioridazine displaying the most cytotoxicity, followed by the piperazinic and aliphatic derivatives. The addition of the phenothiazine derivatives to cell suspensions resulted in significant morphological changes and plasma membrane permeabilization. Octanol/water partition studies revealed that these drugs partitioned preferentially to the apolar phase, even at low pH values (≤4.5). Also, structural and electronic properties were calculated employing density functional theory. Interestingly, the phenothiazine derivatives promoted an immediate dissipation of the mitochondrial transmembrane potential in HTC cells, and the EC50 values were closely correlated with those obtained in cell viability assays, as well as the EC50 for swelling in isolated mitochondria. These results significantly contribute to improving our understanding of the specific structural requirements of the phenothiazine derivatives to induce cell death and suggest the involvement of the mitochondrial permeability transition in phenothiazine-induced cytotoxicity in HTC cells.

Biocatalysis for desymmetrization and resolution of stereocenters beyond the reactive center: How far is far enough?

The kinetic resolution of racemates and desymmetrization are the most common approaches to the preparation of enantiomerically enriched compounds. These procedures allow the access of high valuable, chiral building blocks for many purposes in academic or industrial R&D endeavors. Nevertheless, the scope of stereochemistry recognition in biotransformations usually occurs at the site of the transformation or when it is close to it (not more than 3 bonds). However, there are a growing number of enzymatic transformations which surpass the limits of stereorecognition of remote chiral (or prochiral) centers. In this account, we would like to present some aspects of biocatalyzed remote resolutions and remote desymmetrizations to call attention for these challenging transformations.

Specific calpain activity evaluation in Plasmodium parasites.

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.

In vitro and in vivo activity of an organic tellurium compound on Leishmania (Leishmania) chagasi.

Tellurium compounds have shown several biological properties and recently the leishmanicidal effect of one organotellurane was demonstrated. These findings led us to test the effect of the organotellurium compound RF07 on Leishmania (Leishmania) chagasi, the agent of visceral leishmaniasis in Latin America. In vitro assays were performed in L. (L.) chagasi-infected bone marrow derived macrophages treated with different concentrations of RF07. In in vivo experiments Golden hamsters were infected with L. (L.) chagasi and injected intraperitoneally with RF07 whereas control animals received either Glucantime or PBS. The effect of RF07 on cathepsin B activity of L. (L.) chagasi amastigotes was assayed spectrofluorometrically using fluorogenic substrates. The main findings were: 1) RF07 showed significant leishmanicidal activity against intracellular parasites at submicromolar concentrations (IC50 of 529.7±26.5 nM), and the drug displayed 10-fold less toxicity to macrophages (CC50 of 5,426±272.8 nM); 2) kinetics assays showed an increasing leishmanicidal action of RF07 at longer periods of treatment; 3) one month after intraperitoneal injection of RF07 L. (L.) chagasi-infected hamsters showed a reduction of 99.6% of parasite burden when compared to controls that received PBS; 4) RF07 inhibited the cathepsin B activity of L. (L.) chagasi amastigotes. The present results demonstrated that the tellurium compound RF07 is able to destroy L. (L.) chagasi in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support further study of the potential of RF07 as a possible alternative for the chemotherapy of visceral leishmaniasis.

In vitro and in vivo activity of a palladacycle complex on Leishmania (Leishmania) amazonensis.

BACKGROUND: Antitumor cyclopalladated complexes with low toxicity to laboratory animals have shown leishmanicidal effect. These findings stimulated us to test the leishmanicidal property of one palladacycle compound called DPPE 1.2 on Leishmania (Leishmania) amazonensis, an agent of simple and diffuse forms of cutaneous leishmaniasis in the Amazon region, Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Promastigotes of L. (L.) amazonensis and infected bone marrow-derived macrophages were treated with different concentrations of DPPE 1.2. In in vivo assays foot lesions of L. (L.) amazonensis-infected BALB/c mice were injected subcutaneously with DPPE 1.2 and control animals received either Glucantime or PBS. The effect of DPPE 1.2 on cathepsin B activity of L. (L.) amazonensis amastigotes was assayed spectrofluorometrically by use of fluorogenic substrates. The main findings were: 1) axenic L. (L.) amazonensis promastigotes were destroyed by nanomolar concentrations of DPPE 1.2 (IC50 = 2.13 nM); 2) intracellular parasites were killed by DPPE 1.2 (IC50 = 128.35 nM), and the drug displayed 10-fold less toxicity to macrophages (CC50 = 1,267 nM); 3) one month after intralesional injection of DPPE 1.2 infected BALB/c mice showed a significant decrease of foot lesion size and a reduction of 97% of parasite burdens when compared to controls that received PBS; 4) DPPE 1.2 inhibited the cysteine protease activity of L. (L.) amazonensis amastigotes and more significantly the cathepsin B activity. CONCLUSIONS/SIGNIFICANCE: The present results demonstrated that DPPE 1.2 can destroy L. (L.) amazonensis in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support the potential use of DPPE 1.2 as an alternative choice for the chemotherapy of leishmaniasis.

A new minor dimmeric ester from seeds of Cassia fistula L. (Leguminosae).

Bioactivity-guided fractionation of n-hexane phase from MeOH extract of the seeds of Cassia fistula L. (Leguminosae) yielded two bioactive substances against Cladosporium cladosporioides and C. sphaerospermum. After spectroscopic analysis, these compounds were characterised as the known benzyl 2-hydroxy-3,6-dimethoxybenzoate and its dimer dibenzyl 2,2'-dihydroxy-3,6,3″,6″-tetramethoxy-biphenyl-1,1'-dicarboxylate, which showed a new structural arrangement.

In vitro antileishmanial and antitrypanosomal activities of flavanones from Baccharis retusa DC. (Asteraceae).

Leishmaniasis and Chagas' are parasitic protozoan diseases that affect the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, novel, safe and more efficacious drugs are essential. In this work, the CH(2)Cl(2) phase from MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) was fractioned to afford two flavonoids: naringenin (1) and sakuranetin (2). These compounds were in vitro tested against Leishmania spp. promastigotes and amastigotes and Trypanosoma cruzi trypomastigotes and amastigotes. Compound 2 presented activity against Leishmania (L.) amazonensis, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) chagasi with IC(50) values in the range between 43 and 52 µg/mL and against T. cruzi trypomastigotes (IC(50)=20.17 µg/mL). Despite of the chemical similarity, compound 1 did not show antiparasitic activity. Additionally, compound 2 was subjected to a methylation procedure to give sakuranetin-4'-methyl ether (3), which resulted in an inactive compound against both Leishmania spp. and T. cruzi. The obtained results indicated that the presence of one hydroxyl group at C-4' associated to one methoxyl group at C-7 is important to the antiparasitic activity. Further drug design studies aiming derivatives could be a promising tool for the development of new therapeutic agents for Leishmaniasis and Chagas' disease.