Potential of 2-Hydroxy-3-Phenylsulfanylmethyl-[1,4]-Naphthoquinones against Leishmania (L.) infantum: Biological Activity and Structure-Activity Relationships

Naphtoquinones have been used as promising scaffolds for drug design studies against protozoan parasites. Considering the highly toxic and limited therapeutic arsenal, the global negligence with tropical diseases and the elevated prevalence of co-morbidities especially in developing countries, the parasitic diseases caused by various Leishmania species (leishmaniasis) became a significant public health threat in 98 countries. The aim of this work was the evaluation of antileishmanial in vitro potential of thirty-six 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones obtained by a three component reaction of lawsone, the appropriate aldehyde and thiols adequately substituted, exploiting the in situ generation of o-quinonemethides (o-QM) via the Knoevenagel condensation. The antileishmanial activity of the naphthoquinone derivatives was evaluated against promastigotes and intracellular amastigotes of Leishmania (Leishmania) infantum and their cytotoxicity was verified in mammalian cells. Among the thirty-six compounds, twenty-seven were effective against promastigotes, with IC₅₀ values ranging from 8 to 189 µM; fourteen compounds eliminated the intracellular amastigotes, with IC₅₀ values ranging from 12 to 65 µM. The compounds containing the phenyl groups at R₁ and R₂ and with the fluorine substituent at the phenyl ring at R₂, rendered the most promising activity, demonstrating a selectivity index higher than 15 against amastigotes. A QSAR (quantitative structure activity relationship) analysis yielded insights into general structural requirements for activity of most compounds in the series. Considering the in vitro antileishmanial potential of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones and their structure-activity relationships, novel lead candidates could be exploited in future drug design studies for leishmaniasis.     

Redox-active dinitrodiphenylthioethers against Leishmania: Synthesis,structure–activity relationships and mechanism of action studies

BTB 06237 (2-[(2,4-dichloro-5-methylphenyl)sulfanyl]-1,3-dinitro-5-(trifluoromethyl) benzene), a compound previously identified through QSAR pharmacophore development and a virtual screen of the Maybridge database, possesses potent and selective activity against Leishmania parasites. In the present study, several analogs of BTB 06237 were synthesized and analyzed for activity against Leishmania axenic amastigotes, their ability to reduce the level of parasitemia in peritoneal macrophages, and their ability to generate reactive oxygen species (ROS) in L. donovani promastigotes. It was found that an aromatic ring must be present in the position occupied by the 2,4-dichloro-5-methylphenyl group in the lead compound, but changing the functional groups generally has little effect on the antileishmanial potency. Alterations to the 1,3-dinitro-5-(trifluoromethyl)benzene ring have more influence on antiparasitic activity with two aromatic nitro groups and a third electron-withdrawing group being required. This structural requirement corresponds with redox potential, the ability to generate ROS in the parasites, and dissipation of the mitochondrial membrane potential. Finally, we used this collection of data to design a new antileishmanial compound with strong activity in vitro and improved properties as an antileishmanial candidate.     

Leishmanicidal activity of synthetic antimicrobial peptides in an infection model with human dendritic cells

Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world [14]. The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania [7] and [39]. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis.     

Peganine hydrochloride dihydrate an orally active antileishmanial agent

Protozoic infections caused by genus Leishmania pose an enormous public health threat in developing countries, compounded by the toxicity and resistance to current therapies. Under the aegis of our ongoing program on drug discovery and development on antileishmanial agents from plants, we carried out bioassay guided fractionation on Peganum harmala seeds which resulted in the isolation of 1 as an antileishmanial agent. 2D-NMR spectral data and single crystal X-ray crystallography data indicated 1 as peganine hydrochloride in dihydrated form. The compound 1 exhibited in-vitro activity against both extracellular promastigotes as well as intracellular amastigotes residing within murine macrophages in Leishmania donovani. Furthermore, 1 also exhibited in-vivo activity, 79.6 (±8.07)% against established VL in hamsters at a dose of 100 mg/kg b.wt.     

Synthesis,structural elucidation and in vitro antiparasitic activity against Trypanosoma cruzi and Leishmania chagasi parasites of novel tetrahydro-1-benzazepine derivatives

Forty six new 1,4-epoxy-2-exo-aryl- and cis-2-aryl-4-hydroxytetrahydro-1-benzazepine derivatives were synthesized and fully characterized. All compounds were tested in vitro against both Trypanosoma cruzi and Leishmania chagasi parasites and also for cytotoxicity using Vero and THP-1 mammalian cell lines. Many of the evaluated compounds showed remarkable activity against the epimastigote and intracellular amastigote forms of T. cruzi, with IC₅₀ values comparable with that of control drug nifurtimox, a nitrofuran derivative currently used in the treatment of Chagas’ disease. Other derivatives were found to have good activity against L. chagasi promastigotes, with low toxicity against the mammalian cells, but neither of them was active on intracellular amastigotes of L. chagasi infecting THP-1 macrophages.     

Synthesis,antileishmanial activity and docking study of N′-substitutedbenzylidene-2-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetohydrazides

A series of N′-substitutedbenzylidene-2-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetohydrazide derivatives is synthesized and evaluated for antileishmanial activity against Leishmania donovani promastigotes. Compounds 9a and 9i were shown significant antileishmanial when compared with standard sodium stilbogluconate. Antimicrobial study revealed that compound 9b has potent as well as broad spectrum antibacterial activity when compared with ampicillin and compound 9e showed promising antifungal activity when compared with miconazole. Also, none of the synthesized compounds showed cytotoxicity up to tested concentration. Further, docking study against pteridine reductase 1 enzyme of L. donovani showed good binding interactions. ADME properties of synthesized compounds were also analyzed and showed potential to develop as good oral drug candidates.     

New pyrazolopyrimidine derivatives as Leishmania amazonensis arginase inhibitors

Arginase performs the first enzymatic step in polyamine biosynthesis in Leishmania and represents a promising target for drug development. Polyamines in Leishmania are involved in trypanothione synthesis, which neutralize the oxidative burst of reactive oxygen species (ROS) and nitric oxide (NO) that are produced by host macrophages to kill the parasite. In an attempt to synthesize arginase inhibitors, six 1-phenyl-1H-pyrazolo[3,4-d]pyrimidine derivatives with different substituents at the 4-position of the phenyl group were synthesized. All compounds were initially tested at 100 µM concentration against Leishmania amazonensis ARG (LaARG), showing inhibitory activity ranging from 36 to 74%. Two compounds, 1 (R=H) and 6 (R=CF₃), showed arginase inhibition >70% and IC₅₀ values of 12 µM and 47 µM, respectively. Thus, the kinetics of LaARG inhibition were analyzed for compounds 1 and 6 and revealed that these compounds inhibit the enzyme by an uncompetitive mechanism, showing K_is values, and dissociation constants for ternary complex enzyme-substrate-inhibitor, of 8.5 ± 0.9 µM and 29 ± 5 µM, respectively. Additionally, the molecular docking studies proposed that these two uncompetitive inhibitors interact with different LaARG binding sites, where compound 1 forms more H-bond interactions with the enzyme than compound 6. These compounds showed low activity against L. amazonensis free amastigotes obtained from mice lesions when assayed with as much as 30 µM. The maximum growth inhibition reached was between 20 and 30% after 48 h of incubation. These results suggest that this system can be promising for the design of potential antileishmanial compounds.     

Synthesis and in vitro activity of new tetrahydronaphtho[1,2-b]azepine derivatives against Trypanosoma cruzi and Leishmania chagasi parasites

Series of 2-exo-aryl-1,4-epoxy-2,3,4,5-tetrahydronaphtho[1,2-b]azepines 3a–k and cis-2-aryl-4-hydroxy-2,3,4,5-tetrahydronaphtho[1,2-b]azepines 4a–j were synthesized and evaluated against free and intracellular live forms of Trypanosoma cruzi and Leishmania chagasi parasites using in vitro assays. Cell toxicity was also analyzed on Vero and THP-1 mammalian cell lines. The compounds 3c, 3f, and 4d were the most active against both live forms of T. cruzi parasites with low mammalian cell toxicity. Some compounds were active on free live forms of L. chagasi parasites but none was active on intracellular amastigotes of L. chagasi infecting THP-1 macrophages.     

Pterocarpanquinone LQB-118 Induces Apoptosis in Leishmania (Viannia) braziliensis and Controls Lesions in Infected Hamsters

Previous results demonstrate that the hybrid synthetic pterocarpanquinone LQB-118 presents antileishmanial activity against Leishmania amazonensis in a mouse model. The aim of the present study was to use a hamster model to investigate whether LQB-118 presents antileishmanial activity against Leishmania (Viannia) braziliensis, which is the major Leishmania species related to American tegumentary leishmaniasis. The in vitro antileishmanial activity of LQB-118 on L. braziliensis was tested on the promastigote and intracellular amastigote forms. The cell death induced by LQB-118 in the L. braziliensis promastigotes was analyzed using an annexin V-FITC/PI kit, the oxidative stress was evaluated by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and the ATP content by luminescence. In situ labeling of DNA fragments by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis in the intracellular amastigotes. L. braziliensis-infected hamsters were treated from the seventh day of infection with LQB-118 administered intralesionally (26 µg/kg/day, three times a week) or orally (4,3 mg/kg/day, five times a week) for eight weeks. LQB-118 was active against the L. braziliensis promastigotes and intracellular amastigotes, producing IC50 (50% inhibitory concentration) values of 3,4±0,1 and 7,5±0,8 µM, respectively. LQB-118 induced promastigote phosphatidylserine externalization accompanied by increased reactive oxygen species production and ATP depletion. Intracellular amastigote DNA fragmentation was also observed, without affecting the viability of macrophages. The treatment of L. braziliensis-infected hamsters with LQB-118, either orally or intralesionally, was effective in the control of lesion size, parasite load and increase intradermal reaction to parasite antigen. Taken together, these results show that the antileishmanial effect of LQB-118 extends to L. braziliensis in the hamster model, involves the induction of parasite apoptosis and shows promising therapeutic option by oral or local routes in leishmaniasis.     

Leishmania amazonensis Growth Inhibitors: Biological and Theoretical Features of Sulfonamide 4-Methoxychalcone Derivatives

Current drugs for treating leishmaniasis are still associated with significant toxicity and failure rates. Thus, new effective and less toxic antileishmanial agents are still in need. Herein, we tested a series of sulfonamide 4-methoxychalcone derivatives against L. amazonensis promastigote and amastigote forms to identify its antileishmanial profile against this species compared to L. braziliensis. In addition, we used molecular modeling tools to determine stereoelectronic features that may lead to the antileishmanial profile. Interestingly, all tested compounds were able to affect L. amazonensis promastigote form in a concentration-dependent manner and with low cytotoxicity, except for derivative 3g. However, our results showed that compound 3f (para-Cl) presents the best profile against both L. amazonensis forms (promastigote and amastigote), differently from that observed for L. braziliensis, when compound 3i was the most active. Structure–activity relationship (SAR) analysis of these derivatives pointed molecular volume, HOMO density, and conformational aspects as important characteristics for parasitic profile. Overall, sulfonamide 4-methoxychalcone derivatives may be pointed out not only as lead compounds for treating leishmaniasis (i.e., 3f) but also as experimental tools presenting parasite-selectivity (i.e., 3i).     

The sesquiterpene (−)-α-bisabolol is active against the causative agents of Old World cutaneous leishmaniasis through the induction of mitochondrial-dependent apoptosis

Cutaneous leishmaniasis treatment remains challenging due to the absence of a satisfactory treatment. The screening of natural compounds is a valuable strategy in the search of new drugs against leishmaniasis. The sesquiterpene (?)-α-bisabolol is effective in vivo against visceral leishmaniasis due to Leishmania infantum, but its mechanism of action remains elusive. The aim of this study is to validate this promising compound against the causative species of Old World cutaneous leishmaniasis and to get an insight into its antileishmanial mode of action. The compound was evaluated on L. tropica promastigotes and intracellular amastigotes using bone marrow-derived macrophages and its cytotoxicity was evaluated on L929 fibroblasts. The reactive oxygen species generation was evaluated using a sensitive probe. Mitochondrial depolarization was assessed evaluating the fluorescence due to rhodamine 123 in a flow cytometer. Apoptosis was investigated by measuring the fluorescence due to annexin V and propidium iodide in a flow cytometer. The ultrastructure of treated promastigotes and intracellular amastigotes was analysed through transmission electron microscopy. (?)-α-Bisabolol was active against L. tropica intracellular amastigotes displaying an inhibitory concentration 50 % of 25.2 µM and showing low cytotoxicity. This compound induced time and dose-dependent oxidative stress, mitochondrial depolarization and phosphatidilserine externalization (a marker of apoptosis). These effects were noticed at a low concentration and short exposure time. In the ultrastructural analyses, the treated parasites showed mitochondrial disruption, presence of electron-dense structures and chromatin condensation. These results suggest that this natural compound induces oxidative stress and mitochondrial-dependent apoptosis on Leishmania without disturbing the plasma membrane.     

Antileishmanial activity and ultrastructural changes of sesquiterpene lactones isolated from Calea pinnatifida (Asteraceae)

Bioactivity-guided fractionation of antileishmanial active CH₂Cl₂ phase of MeOH extract from leaves of Calea pinnatifida led to isolation of two sesquiterpene lactones calein C (1) and calealactone C (2), which structures were stablished on the basis of spectroscopic analysis. Compounds 1 and 2 displayed potent activity against Leishmania amazonensis promastigotes with EC₅₀ of 1.7 and 4.6 µg mL⁻¹, respectively. Compound 2 presented low cytotoxicity for J774 macrophages and displayed activity against amastigote forms of L. amazonensis similar to miltefosine with CC₅₀ values of 31.73 and 27.18 µg mL⁻¹, respectively. Additionally, compounds 1 and 2 caused ultrastructural changes in promastigotes leading to a loss of their classical structural morphology, as evidenced by electron microscopy. Also compound 2 decreased the mitochondria membrane potential. To the best of our knowledge, this is the first occurrence of 1 and 2 in C. pinnatifida. The results obtained highlighted the importance of studying sesquiterpene lactones isolated from Calea pinnatifida in terms of antileishmanial activity, in order to understand the mechanism of action of the isolated compounds in promastigotes forms of L. amazonensis.     

Targeting the human parasite Leishmania donovani: Discovery of a new promising anti-infectious pharmacophore in 3-nitroimidazo[1,2-a]pyridine series

We report herein the discovery of antileishmanial molecules based on the imidazo[1,2-a]pyridine ring. In vitro screenings of imidazopyridines belonging to our chemical library, toward the promastigotes stage of Leishmania donovani, J774A.1 murine and HepG2 human cells, permitted to identify three selective hit-compounds (12, 20 and 28). New derivatives were then synthesized to allow structure–activity and –toxicity relationships analyses, enabling to characterize a lead-compound (44) displaying both a high potency (IC₅₀ = 1.8 μM) and a good selectivity index, in comparison with three antileishmanial reference drug-compounds (amphotericin B, miltefosine and pentamidine). Moreover, lead-compound 44 also exhibits good in vitro activity against the intracellular amastigote stage of L. donovani. Thus, the 6-halo-3-nitro-2-(phenylsulfonylmethyl)imidazo[1,2-a]pyridine scaffold appears as a new promising selective antileishmanial pharmacophore, especially when substituted at position 8 by a bromine atom.     

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC₅₀ = 28.5 ± 2.0 μM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.     

High throughput screens yield small molecule inhibitors of Leishmania CRK3:CYC6 cyclin-dependent kinase

Background

Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.

Methodology/Principal Findings

In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.

Conclusions/Significance

The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.     

Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice

Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨ_m) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches.     

Antileishmanial activity of new thiophene–indole hybrids: Design,synthesis, biological and cytotoxic evaluation,and chemometric studies

In the present work, thirty-two hybrid compounds containing cycloalka[b]thiophene and indole moieties (TN5, TN5 1–7, TN6, TN6 1–7, TN7, TN7 1–7, TN8, TN8 1–7) were designed, synthesized and evaluated for their cytotoxic and antileishmanial activity against Leishmania amazonensis promastigotes. More than half of the compounds (18 compounds) exhibited significant antileishmanial activity (IC₅₀ lower than 10.0 μg/L), showing better performance than the reference drugs (tri- and penta-valent antimonials). The most active compounds were TN8-7, TN6-1 and TN7 with respective IC₅₀ values of 2.1, 2.3 and 3.2 μg/mL. Demonstrating that all of the compounds were less toxic than the reference drugs, even at the highest evaluated concentration (400 μg/mL), no compound tested presented human erythrocyte cytotoxicity. Compound TN8-7’s effectiveness against a trivalent antimony-resistant culture was demonstrated. It was observed that TN8-7’s antileishmanial activity is associated with DNA fragmentation of L. amazonensis promastigotes. Chemometric studies (CPCA, PCA, and PLS) highlight intrinsic solubility/lipophilicity, and compound size and shape as closely related to activity. Our results suggest that hybrid cycloalka[b]thiophene–indole derivatives may be considered as lead compounds for further development of new drugs for the treatment of leishmaniasis.     

In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites

Background

The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro.

Methodology/Principal Findings

We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance.

Conclusions/Significance

Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.     

Synthesis and activity of novel tetrazole compounds and their pyrazole-4-carbonitrile precursors against Leishmania spp

A new series of 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazole derivatives (4a–m) and their precursor 1-aryl-3-methyl-1H-pyrazole-4-carbonitriles (3a–m) were synthesized and evaluated as antileishmanials against Leishmania braziliensis and Leishmania amazonensis promastigotes in vitro. In parallel, the cytotoxicity of these compounds was evaluated on the RAW 264.7 cell line. The results showed that among the assayed compounds the substituted 3-chlorophenyl (4a) (IC₅₀/24 h = 15 ± 0.14 μM) and 3,4-dichlorophenyl tetrazoles (4d) (IC₅₀/24 h = 26 ± 0.09 μM) were the most potent against L. braziliensis promastigotes, as compared the reference drug pentamidine, which presented IC₅₀ = 13 ± 0.04 μM. In addition, 4a and 4d derivatives were less cytotoxic than pentamidine. However, these tetrazole derivatives (4) and pyrazole-4-carbonitriles precursors (3) differ against each of the tested species and were more effective against L.braziliensis than on L. amazonensis.     

Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro

Tamoxifen is an antagonist of the estrogen receptor and currently used for the treatment of breast cancer. The current treatment of cutaneous leishmaniasis with pentavalent antimony compounds is not satisfactory. Therefore, in this study, due to its antileishmanial activity, effects of tamoxifen on the growth of promastigotes and amastigotes of Leishmania major Iranian strain were evaluated in vitro. Promastigotes and amastigotes were treated with different concentrations (1, 5, 10, 20, and 50 μg/ml) and time periods (24, 48, and 72 hr) of tamoxifen. After tamoxifen treatment, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 biphenyl tetrazolium bromide assay) was used to determine the percentage of live parasites and Graph Pad Prism software to calculate IC₅₀. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration (IC₅₀) of tamoxifen on promastigotes was 2.6 μg/ml after 24 hr treatment. Flow cytometry analysis showed that tamoxifen induced early and late apoptosis in Leishmania promastigotes. While after 48 hr in control group the apoptosis was 2.0%, the 50 µg/L concentration of tamoxifen increased it to 59.7%. Based on the in vitro antileishmanial effect, tamoxifen might be used for leishmaniasis treatment; however, further researches on in vivo effects of tamoxifen in animal models are needed.