Cell Line J774 Research Articles

Organically Modified Silica Nanoparticles Interaction with Macrophage Cells: Assessment of Cell Viability on the Basis of Physicochemical Properties

Silica nanoparticles have drawn a lot of attention for nanomedicine application, and this is attributed to their biocompatibility and ease of surface functionalization. However, successful utilization of these inorganic systems for biomedical application depends on their physicochemical properties. This study, therefore, discusses in vitro toxicity of organically modified silica nanoparticles on the basis of size, shape, and surface properties of silica nanoparticles. Spherical- and oval-shaped nanoparticles having hydroxyl and amine groups were synthesized in Tween 80 micelles using different organosilanes. Nanoparticles of similar size and morphology were considered for comparative assessment. "As-prepared" nanoparticles were characterized in terms of size, shape, and surface properties using ZetaSizer, transmission electron microscopy, and Fourier transform infrared to establish the above parameters. In vitro analysis in terms of nanoparticle-based toxicity was performed on J-774 (macrophage) cell line using propidium iodide-4',6-diamidino-2-phenylindol and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Fluorescent dye-entrapped nanoparticles were used to visualize the uptake of the nanoparticles by macrophage cells. Results from cell studies suggested low levels of toxicity for different nanoparticle formulations studied, therefore are suitable for nanocarrier application for poorly soluble molecules. On the contrary, the nanoparticles of similar size and shape, having amine groups and low net negative charge, do not exhibit any in vitro cytotoxicity.

Evaluation of in vitro anti-inflammatory effects of crude ginger and rosemary extracts obtained through supercritical CO2 extraction on macrophage and tumor cell line: the influence of vehicle type.

BackgroundNumerous plants from have been investigated due to their anti-inflammatory activity and, among then, extracts or components of ginger (Zingiber officinale Roscoe) and rosemary (Rosmarinus officinalis L.), sources of polyphenolic compounds. 6-gingerol from ginger rhizome and carnosic acid and carnosol from rosemary leaves present anti-tumor, anti-inflammatory and antioxidant activities. However, the evaluation of the mechanisms of action of these and other plant extracts is limited due to their high hydrophobicity. Dimethylsulfoxide (DMSO) is commonly used as a vehicle of liposoluble materials to mammalian cells in vitro, presenting enhanced cell penetration. Liposomes are also able to efficiently deliver agents to mammalian cells, being capable to incorporate in their structure not only hydrophobic molecules, but also hydrophilic and amphiphilic compounds. Another strategy is based on the use of Pluronic F-68, a biocompatible low-foaming, non-ionic surfactant, to disperse hydrophobic components. Here, these three delivery approaches were compared to analyze their influence on the in vitro anti-inflammatory effects of ginger and rosemary extracts, at different concentrations, on primary mammalian cells and on a tumor cell line.MethodsGinger and rosemary extracts free of organic solvents were obtained by supercritical fluid extraction and dispersed in DMSO, Pluronic F-68 or liposomes, in variable concentrations. Cell viability, production of inflammatory mediators and nitric oxide (NO) release were measured in vitro on J774 cell line and murine macrophages primary culture stimulated with bacterial lipopolysaccharide and interferon-γ after being exposed or not to these extracts.ResultsGinger and rosemary extracts obtained by supercritical CO2 extraction inhibited the production of pro-inflammatory cytokines and the release of NO by peritoneal macrophages and J774 cells. The delivery vehicles influenced the anti-inflammatory effects. Comparatively, the ginger extract showed the highest anti-inflammatory activity on the tumor cell line. Controversially, rosemary extract dispersed on DMSO induced a more significant IL-1 and TNF-α reduction than ginger extract in primary macrophages.ConclusionsAmongst the tested delivery vehicles, DMSO was the most suitable, presenting reduced cytotoxicity, followed by Pluronic F-68 and liposomes, provably due to differences in their form of absorption, distribution and cellular metabolism. Co-administration of liposomes and plant extracts may cause death of macrophages cells and induction of NO production. It can be concluded that some of the beneficial effects attributed to extracts of ginger and rosemary may be associated with the inhibition of inflammatory mediators due to their high antioxidant activity. However, these effects were influenced by the type of delivery vehicle.

Antibacterial and Antiparasitic Activity of Manganese(I) Tricarbonyl Complexes with Ketoconazole, Miconazole, and Clotrimazole Ligands

Five manganese(I) tricarbonyl complexes of the general formula [Mn(CO)3(bpyR,R)(azole)]PF6 with R = H, COOCH3, and azole = ketoconazole (ktz), miconazole (mcz), and clotrimazole (ctz) were synthesized and fully charaterized, including X-ray structure analysis for the ctz compound. The antibacterial activity on a panel of eight Gram-positive and Gram-negative bacterial strains was determined. While there was no effect on the latter microorganisms, the ctz complex showed submicromolar activity on Staphylococcus aureus and S. epidermidis with MIC values of 0.625 μM. Antiparasitic activity was investigated on Leishmania major and Trypanosoma brucei. Coordination of the organic azole drugs to the Mn(CO)3 moiety led to complexes with low micromolar IC50 values, but their potential for antileishmanial therapy is low due to comparable toxicity on mammalian cell lines 293T and J774.1. In contrast, the antitrypanosomal activity is much more promising, and the most potent compound incorparting the ktz ligand has an ...

Organic Solvent-Free, One-Step Engineering of Graphene-Based Magnetic-Responsive Hybrids Using Design of Experiment-Driven Mechanochemistry.

In this study, we propose an organic solvent-free, one-step mechanochemistry approach to engineer water-dispersible graphene oxide/superparamagnetic iron oxide (GO/SPIOs) hybrids, for biomedical applications. Although mechanochemistry has been proposed in the graphene field for applications such as drug loading, exfoliation or polymer-composite formation, this is the first study to report mechanochemistry for preparation of GO/SPIOs hybrids. The statistical design of experiment (DoE) was employed to control the process parameters. DoE has been used to control formulation processes of other types of nanomaterials. The implementation of DoE for controlling the formulation processes of graphene-based nanomaterials is, however, novel. DoE approach could be of advantage as one can tailor GO-based hybrids of predicted yields and compositions. Hybrids were characterized by TEM, AFM FT-IR, Raman spectroscopy, and TGA. The dose–response magnetic resonance (MR) properties were confirmed by MR imaging of phantoms. The biocompatibility of the hybrids with A549 and J774 cell lines was confirmed by the modified LDH assay.

Mg-doped biphasic calcium phosphate by a solid state reaction route: Characterization and evaluation of cytotoxicity

Hydroxyapatite (HAP) and β-tricalcium phosphate (β-TCP) are widely used in tissue engineering because of their chemical similarity to the inorganic bone phase. In this work, we prepare biphasic calcium phosphate (BCP, a mixture of HAP and β-TCP) doped with different concentrations of magnesium to investigate the influence of magnesium on the BCP crystal structure. Magnesium is known to be an important element in the composition of bones and teeth. Recent research has shown that the doping of magnesium into BCP improves its bone metabolism and mechanical properties without affecting its biocompatibility. The samples were prepared by solid-state reaction from calcium carbonate, monobasic ammonium phosphate, and magnesium nitrate hexahydrate. Varying concentrations of magnesium were used and its modifications were examined by different characterization techniques. The phase composition and morphology of the ceramic powders were characterized by X-ray diffraction and scanning electron microscopy, respectively. The functional groups were analyzed using Fourier transform infrared spectroscopy and Raman spectroscopy. Cell viability experiments, using macrophage-like cell lines J774, showed that the synthesized Mg-doped BCP did not exhibit cytotoxicity regardless of the doses assayed or the different concentrations of magnesium used, suggesting it as a good material for potential biological applications.

Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells.

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc2155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc2155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc2155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.

CD13 mediates phagocytosis in human monocytic cells

The myelomonocytic marker aminopeptidase N/CD13 is a novel phagocytic receptor in monocytes and macrophages.

Effect of SXWS/WSXWS peptides on chemotaxis and adhesion of the macrophage-like cell line J774.

WSXWS motif is a conserved amino acid sequence that is present in type I cytokine receptors. This motif that can be found both in the ligand binding chains and signal transducer molecule of the receptors with different amino acids at the position "X" plays a role in the receptor folding, ligand binding and signal transduction as well. Structural analysis proved that WSEWS motif of IL-6R is located in a highly accessible location in the protein. Structural properties and chemotaxis of a tetrapeptide library with SXWS sequence, where X was the 19 proteinogenic amino acids except cystein were systematically studied earlier. It has been proved that C-terminal amidation and the identity of amino acid X had a pronounced influence on the chemotactic properties but less of the structure of the peptides. Here, we present our findings on the effect of a tetrapeptide and a pentapeptide library with the sequence of SXWS and WSXWS on the chemotaxis and adhesion of J774 murine macrophage cell line. We studied the effect of the presence/absence of N-terminal tryptophan and the different amino acids at the X position on these physiological responses. Results indicated that amino acid X had a marked influence on chemotaxis, adhesion as well as on proliferation induced by (W)SXWS peptides. Elongation of SXWS sequence with a tryptophan at the N terminus also altered pronouncedly all the physiological responses of the cells studied. A good correlation could be observed between the chemotaxis and the proliferation and physicochemical parameters of the amino acid X.

Fruit Extract from A Sechium edule Hybrid Induce Apoptosis in Leukaemic Cell Lines but not in Normal Cells

The antiproliferative potential of a crude extract from the chayote hybrid H-837-07-GISeM® and its potential for apoptosis induction were assessed in leukaemic cell lines and normal mouse bone marrow mononuclear cells (BM-MNCs). The extract strongly inhibited the proliferation of the P388, J774, and WEHI-3 cell lines (with an IC50 below 1.3 μg·mL−1), reduced cell viability, and induced apoptotic body production, phosphatidylserine translocation, and DNA fragmentation. However, the extract had no effect on BM-MNCs. We postulate that these properties make the extract a good candidate for an anti-tumour agent for clinical use.

Heterologous Expression of 3-O-Deacylase in Acinetobacter baumannii Modulates the Endotoxicity of Lipopolysaccharide

The lipopolysaccharide (LPS) of Acinetobacter baumannii is a potent stimulator of proinflammatory cytokines, such as interleukin-6 (IL-6). The 3-O-deacylase (PagL)-modifying enzyme that removes the 3-O-linked acyl chain from the disaccharide backbone of lipid A provides the opportunity to develop a new therapeutic compound that could reduce detrimental inflammatory responses. The plasmid pMMB66EH-PagL obtained by recombinant DNA technology was electroporated into A. baumannii ATCC 19606. Compared with wild-type LPS, outer membrane vesicles and inactivated whole cells of engineered bacteria had a statistically significant decreased ability to produce IL-6. Structural analysis of lipid A by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that the profile of lipid A fractions under PagL expression was changed. Taken together, our data showed that recombinant penta-acylated lipid A had less immunoreactivity and that the tetra-acylated version of lipid A with TLR4 antagonist activity decreased the induction of IL-6 production in the murine macrophage cell line J774 A.1.

Nitric oxide is an important regulator of heme oxygenase-1 expression in the lipopolysaccharide and interferon-γ-treated murine macrophage-like cell line J774.1/JA-4.

Heme oxygenase-1 (HO-1) catabolizes the degradation of heme into bilirubin, carbon monoxide, and iron ions. The HO-1 products provide antioxidant cytoprotection in addition to having potent antiinflammatory and immunomodulatory functions. HO-1 is induced by its substrate heme and environmental factors including oxidative and heat stresses. Although previous studies reported that lipopolysaccharide (LPS) induced the expression of both the HO-1 gene and its protein in macrophages, the major regulators of HO-1 expression remain unknown. To identify these regulators, we used two types of cell, the murine macrophage-like cell line J774.1/JA-4 and its LPS-resistant mutant, LPS1916. Based on a comparison of the results obtained with these cells, we found that nitric oxide (NO) was closely linked to the induction of HO-1. Real-time polymerase chain reaction (PCR) showed that the time course for inducible HO-1 mRNA by LPS or LPS+interferon (IFN)-γ was similar to that for inducible NO synthase (iNOS) mRNA. Furthermore, the expression of iNOS mRNA and protein increased earlier than that of HO-1 mRNA and protein. N-Nitro-L-arginine methyl ester, an NO synthase inhibitor, reduced both HO-1 expression and NO production in LPS+IFN-γ-treated JA-4 cells. Furthermore, NOC-12, an NO donor, significantly induced HO-1 expression not only in JA-4 but also in LPS1916 cells. Reactive oxygen species (ROS) scavengers, such as superoxide dismutase and catalase, did not affect HO-1 protein expression in LPS+IFN-γ-treated JA-4 cells. These results suggest that, among ROS, NO plays an important role in HO-1 induction in activated macrophages treated with LPS+IFN-γ.

THE USE OF NITRIC OXIDE FREE RADICAL SENSITIVE FLUOROPHORES TO DETECT MACROPHAGE PHAGOLYSOSOME ACTIVITY

Macrophages are phagocytes which facilitate innate immunity via phagocytosis, averting antagonistic effects resulting from bacterial infections. This is a strictly choreographed event initiated by bacterial-macrophage interactions between pathogen associated molecular patterns and toll-like receptors in macrophages. Consequently, the pathogen is ingested by the macrophage through a vacuole which matures to obtain an arsenal of antimicrobial properties including nitric oxide free-radicals (NO∙). Inducible nitric oxide synthase is an enzyme accountable for NO∙ production upon stimulation. This study utilized opportunistic pathogens Staphylococcus epidermidis, Serratia marcescens, an assemblage composed of the two species and a Lipopolysaccharide positive control to challenge the murine-macrophage J774 Cell-line. Phagolysosome activity was assessed using NO∙ sensitive fluorophore, DAF-FMDA. Fluorescence activity was measured for 300 seconds using a Nikon Eclipse TE200 fluorescence microscope and DXM1200F camera. In all treatments, maximal fluorophore activity was attained within 20 seconds; level of fluorophore activity was dependent on the treatment. S. epidermidis and the bacterial assemblage initiated relatively high activities (RFU = 73.48 ± 3.52 SD; RFU = 56.66 ± 4.74 respectively), comparable to the positive control (RFU = 71.66 ± 0.90). S. marcescens induction of fluorophore activity occurred, but to a lesser extent (RFU = 48.72 ± 3.36), over 20 seconds. The current study suggests the Gram positive S. epidermidis incites relatively high levels of NO∙ synthesis similar to the positive control which was primed with commercial Lipopolysaccharide in challenged macrophages while the NO∙ levels induced by Gram negative S. marcescens were inferior. The response to the bacterial assemblage largely mimicked the response to S. epidermidis alone suggesting macrophages preferentially phagocytosed this species. This study suggests that immune responses by macrophages depend on the bacterial species and therefore does not follow a consistent pattern, particularly in short term NO∙ synthesis.

Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis.

Design, synthesis and biological evaluation of novel 4-alkapolyenylpyrrolo[1,2-a]quinoxalines as antileishmanial agents – Part III

A series of new 4-alkapolyenylpyrrolo[1,2-a]quinoxaline derivatives, original and structural analogues of alkaloid chimanine B and of previously described 4-alkenylpyrrolo[1,2-a]quinoxalines, was synthesized in good yields using efficient palladium-catalyzed Suzuki–Miyaura cross-coupling reactions. These new compounds were tested for in vitro antiparasitic activity upon three Leishmania spp. strains. Biological results showed activity against the promastigote forms of L. major, L. mexicana and L. donovani with IC50 ranging from 1.2 to 14.7 μM. In attempting to investigate if our pyrrolo[1,2-a]quinoxaline derivatives are broad-spectrum antiprotozoal compounds activities toward one Trypanosoma brucei brucei strain and the W2 and 3D7 Plasmodium falciparum strains were also investigated. In parallel, the in vitro cytotoxicity of these molecules was assessed on the murine J774 and human HepG2 cell lines. Structure–activity relationships of these new synthetic compounds are here discussed.

Effects of strains of Lactococcus lactis on the production of nitric oxide and cytokines in murine macrophages.

Nitric oxide (NO) is a multifunctional mediator that is involved in a variety of pathologic and physiologic processes. Few studies have addressed the effect of lactic acid bacteria (LAB), especially Lactococcus lactis strains used in dairy products, on inducible nitric oxide synthase (iNOS) induction as a component of the host's gastrointestinal immune response. We investigated the ability of L. lactis strains to induce NO synthesis in the murine macrophage-like cell line J774.1 and in peritoneal macrophages from mice. The degree of NO induction was specific to the L. lactis strain used. Compared with the no-treatment control, heat treatment of L. lactis cells decreased NO and TNF-α levels but further stimulated interleukin (IL)-12 production. Adding L. lactis cells to peritoneal macrophages dose-dependently increased the production of NO and IL-10 but decreased that of IL-12p70. Adding L. lactis cells to interferon-γ-stimulated J774.1 cells enhanced cell death and the production of NO and IL-12p40, whereas addition of 1400W, a specific inhibitor of iNOS, decreased NO production and cell death. Conversely, adding 1400W to J774.1 cells further enhanced IL-12p40 production, suggesting that IL-12 production is perturbed by excess endogenous NO. IL-12 production is thought to be a marker of improved immunostimulation. Our results suggest that IL-12 production could be increased by limiting endogenous NO production.

A role for LHC1 in higher order structure and complement binding of the Cryptococcus neoformans capsule.

Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus Cryptococcus neoformans. In the present study, we demonstrate an unusual role for a secreted lactonohydrolase of C. neoformans, LHC1 in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and lhc1Δ strains by dynamic and static light scattering suggested a role for the LHC1 locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in LHC1-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen.

Microevolution During Serial Mouse Passage Demonstrates FRE3 as a Virulence Adaptation Gene in Cryptococcus neoformans

ABSTRACTPassage in mice of opportunistic pathogens such as Cryptococcus neoformans is known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype A C. neoformans identified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of a FRE3-encoded iron reductase in the two strains but not in a control strain that did not demonstrate increased virulence during mouse passage. FRE3 was shown to express an iron reductase activity and to play a role in iron-dependent growth of C. neoformans. Overexpression of FRE3 in the two original environmental strains increased growth in the macrophage cell line J774.16 and increased virulence. These data demonstrate a role for FRE3 in the virulence of C. neoformans and demonstrate how the increased expression of such a “virulence acquisition gene” during the environment-to-mammal transition, can optimize the virulence of environmental strains in mammalian hosts.

Antineoplastic activity of rinvanil and phenylacetylrinvanil in leukaemia cell lines.

In the search for novel chemotherapeutic agents for cancer treatment, capsaicin has been shown to inhibit proliferation and induce apoptosis in various types of cancer cell line, including leukaemia cell lines. The capsaicin analogues, rinvanil and phenylacetylrinvanil (PhAR), share a binding affinity for vanilloid receptors and may have biological activities similar to capsaicin; however, their anticancer potential has not yet been reported. This study analyses the antineoplastic activities of rinvanil and PhAR in leukaemia versus normal cells. P388, J774 and WEHI-3 leukaemia cell lines, as well as mouse bone marrow mononuclear cells, were cultured with varying concentrations of rinvanil and PhAR. Following this, proliferation and apoptosis were determined by the sulforhodamine B (SRB) assay and DNA ladder. Cultured leukaemia cell lines and mouse bone marrow mononuclear cells demonstrated a dose-dependent inhibition of proliferation, while non-diseased cells were less sensitive to the cytotoxic effect of capsaicin, rinvanil and PhAR. Rinvanil and PhAR also induced apoptosis in leukaemia cell lines but not in bone marrow. Given the lower IC50 values for apoptosis induction in leukaemia cells compared with that of normal cells, PhAR is a promising selective anticancer agent.

Suspension culture of Besnoitia caprae by murine macrophage.

Besnoitia caprae is a tissue cyst-forming protozoan that infects goats and has considerable economic importance in certain regions of Asia and Africa. Murine macrophage J774 cell line was inoculated with tachyzoites of Besnoitia caprae (BC-Pars isolate) collected from mice. A significant growth of tachyzoites was observed in J774. Mice were inoculated with tachyzoites harvested from J774 cell culture. Skin samples from the mice infected with tachyzoites of BC-Pars were PCR positive. One mouse showed alopecia and skin lesions on 45 DPI. Dermal lesions started from around right eye and gradually developed more and more. After euthanasia on 60 DPI, histopathological evaluation of skins around the eye showed necrosis of the epidermis and follicular adnexa with chronic inflammatory cell infiltration. Histopathological sections of their skin showed the presence of necrosis and mononuclear cell infiltration. To the authors' knowledge, this is the first report of successful production of Besnoitia caprae tachyzoites was achieved in vitro by suspension culture technique. Another interesting finding is the report of the alopecia and skin lesions around the eye in mouse that quite similar to lesions of goats due to infection of Besnoitia caprae.

Antileishmanial Lead Structures from Nature: Analysis of Structure-Activity Relationships of a Compound Library Derived from Caffeic Acid Bornyl Ester

Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii (V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester (1) as the active component against Leishmania major (L. major) promastigotes (IC50 = 48.8 µM). To investigate the structure-activity relationship (SAR), a library of compounds based on 1 was synthesized and tested in vitro against L. major and L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some compounds showed antileishmanial activity in the concentration range of pentamidine and miltefosine which are the standard drugs in use. In the L. major amastigote assay compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM. The Michael system seems not to be essential for antileishmanial activity. Based on the results compound 27 can be regarded as new lead structure for further structure optimization.