The mesoionic compound MI-D changes energy metabolism and induces apoptosis in T98G glioma cells.

The mesoionic compound 4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride (MI-D) impairs mitochondrial oxidative phosphorylation and has a significant antitumour effect against hepatocarcinoma and melanoma. This study evaluated the cytotoxic effect of MI-D on T98G glioblastoma cells and investigated whether the impairment of oxidative phosphorylation promoted by MI-D is relevant to its cytotoxic effect. The effects of MI-D on T98G cells cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) HG (glycolysis-dependent) and galactose plus glutamine-supplemented Dulbecco's modified Eagle's medium (DMEM) GAL (oxidative phosphorylation-dependent) were compared. T98G cells grown in DMEM GAL medium exhibited higher respiration rates and citrate synthase activity and lower lactate levels, confirming the metabolic shift to oxidative phosphorylation in these cells. MI-D significantly decreased the cell viability in a dose-dependent manner in both media; however, T98G cells cultured in DMEM GAL medium were more susceptible. The mesoionic significantly inhibited mitochondrial oxidative phosphorylation of glioma cells in both media. At the same time, lactate levels were not altered, indicating an absence of compensatory glycolysis activation. Additionally, MI-D increased the citrate synthase activity of cells in both media, which in DMEM HG-cultivated cells was followed by citrate accumulation. Apoptosis dependent on caspase-3 mediated the toxicity of MI-D on T98G cells. The higher susceptibility of glioma cells cultured in DMEM GAL medium to MI-D indicates that the impairment of mitochondrial functions is involved in mesoionic cytotoxicity. The results of this study indicate the potential use of MI-D for glioblastoma treatment.

Fluorine Atoms on C6H5-Corrole Affect the Interaction with Mpro and PLpro Proteases of SARS-CoV-2: Molecular Docking and 2D-QSAR Approaches.

The chymotrypsin-like cysteine protease (3CLpro, also known as main protease-Mpro) and papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been used as the main targets for screening potential synthetic inhibitors for posterior in vitro evaluation of the most promising compounds. In this sense, the present work reports for the first time the evaluation of the interaction between Mpro/PLpro with a series of 17 porphyrin analogues-corrole (C1), meso-aryl-corrole (C2), and 15 fluorinated-meso-aryl-corrole derivatives (C3-C17) via molecular docking calculations. The impact of fluorine atoms on meso-aryl-corrole structure was also evaluated in terms of binding affinity and physical-chemical properties by two-dimensional quantitative structure-activity relationship (2D-QSAR). The presence of phenyl moieties increased the binding capacity of corrole for both proteases and depending on the position of fluorine atoms might impact positively or negatively the binding capacity. For Mpro the para-fluorine atoms might decrease drastically the binding capacity, while for PLpro there was a certain increase in the binding affinity of fluorinated-corroles with the increase of fluorine atoms into meso-aryl-corrole structure mainly from tri-fluorinated insertions. The 2D-QSAR models indicated two separated regions of higher and lower affinity for Mpro:C1-C17 based on dual electronic parameters (σI and σR), as well as one model was obtained with a correlation between the docking score value of Mpro:C2-C17 and the corresponding 13C nuclear magnetic resonance (NMR) chemical shifts of the sp2 carbon atoms (δC-1 and δC-2) of C2-C17. Overall, the fluorinated-meso-aryl-corrole derivatives showed favorable in silico parameters as potential synthetic compounds for future in vitro assays on the inhibition of SARS-CoV-2 replication.

Synthetic (E)-3-Phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium Chloride Derivatives as Promising Chemotherapy Agents on Cell Lines Infected with HTLV-1.

Synthesis of four compounds belonging to mesoionic class, (E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride derivatives (5a-d) and their biological evaluation against MT2 and C92 cell lines infected with human T-cell lymphotropic virus type-1 (HTLV-1), which causes adult T-cell leukemia/lymphoma (ATLL), and non-infected cell lines (Jurkat) are reported. The compounds were obtained by convergent synthesis under microwave irradiation and the cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Results showed IC50 values of all compounds in the range of 1.51-7.70 M in HTLV-1-infected and non-infected cells. Furthermore, it was observed that 5b could induce necrosis after 24 h for Jurkat and MT2 cell lines. The experimental (fluorimetric method) and theoretical (molecular docking) results suggested that the mechanism of action for 5b could be related to its capacity to intercalate into DNA. Moreover, the preliminary pharmacokinetic profile of the studied compounds (5a-d) was obtained through human serum albumin (HSA) binding affinity using multiple spectroscopic techniques (circular dichroism, steady-state and time-resolved fluorescence), zeta potential and molecular docking calculations. The interaction HSA:5a-d is spontaneous and moderate (Ka ~ 104 M-1) via a ground-state association, without significantly perturbing both the secondary and surface structures of the albumin in the subdomain IIA (site I), indicating feasible biodistribution in the human bloodstream.

In vitro and in vivo cytotoxic activity and human serum albumin interaction for a methoxy-styryl-thiosemicarbazone.

Thiosemicarbazone is a class of compounds with potential applications in medicine, presenting high capacity to inhibit the growth of cancer cells as well as low toxicity. Because of high interest in anticancer studies involving thiosemicarbazones as new chemotherapeutic agents, a synthetic thiosemicarbazone derivative, 4-N-(2'-methoxy-styryl)-thiosemicarbazone (MTSC) was evaluated in vivo against Ehrlich carcinoma in an animal model. In vivo results demonstrated that MTSC treatment induced the survival of mice and altered significantly the body weight of the surviving mice 12 days after tumor inoculation. Treatment with 30 mg/kg of MTSC exhibited effective cytotoxic activity with T/C values of 150.49% (1 dose) and 278% (2 doses). Its interaction with human serum albumin (HSA), which plays a crucial role in the biodistribution of a wide variety of ligands, was investigated by multiple spectroscopic techniques at 296 K, 303 K, and 310 K, as well as by theoretical calculations. The interaction between HSA and MTSC occurs via ground-state association in the subdomain IIA (Sudlow's site I). The binding is moderate (Ka ≈ 104 M-1), spontaneous, entropically, and enthalpically driven. Molecular docking results suggested hydrogen bonding and hydrophobic interactions as the main binding forces. Overall, the interaction HSA:MTSC could provide therapeutic benefits, improving its cytotoxic efficacy and tolerability.

Synthesis and biological evaluation of N-aryl-2-phenyl-hydrazinecarbothioamides: Experimental and theoretical analysis on tyrosinase inhibition and interaction with HSA.

A series of N-aryl-2-phenyl-hydrazinecarbothioamides have been investigated as possible inhibitors of tyrosinase, an enzyme involved in the development of melanomas. The hydrazinecarbothioamides 1-6 were synthesized from the reaction between phenylhydrazine and isothiocyanates, for which three different methods have been employed, namely stirring at room temperature, by microwave irradiation or by mechanochemical grinding. Quantitative yields were obtained for the later technique. Compound 4 showed the best value for tyrosinase inhibition (IC50 = 22.6 µM), which occurs through an uncompetitive mechanism. Molecular docking results suggested that 4 can interact via T-stacking with the substrate L-DOPA and via hydrogen bonding and hydrophobic forces with the amino acid residues Ala-79, His-243, Val-247, Phe-263, Val-282, and Glu-321. The interaction between human serum albumin (HSA) and compound 4 occurs through a ground state association and does not perturb the secondary structure of the albumin as well as the microenvironment around Tyr and Trp residues. The binding is spontaneous, moderate and occurs mainly in the Sudlow's site I. Molecular docking results suggested hydrogen bonding, hydrophobic and electrostatic interactions as the main binding forces between the compound 4 and the amino acid residues Lys-198, Trp-214, Glu-449, Leu-452, and Leu-480.

Thiosemicarbazones as inhibitors of tyrosinase enzyme.

In the search for compounds which may inhibit the development of melanomas, a series of thiosemicarbazones has been investigated as possible inhibitors of the tyrosinase enzyme. The results showed that all the thiosemicarbazones tested exhibited significant inhibitory effects on the enzyme. Thiosemicarbazones Thio-1, Thio-2, Thio-3 and Thio-4 substituted with oxygenate moieties, were better inhibitors (IC50 0.42, 0.35, 0.36 and 0.44mM, respectively) than Thio-5, Thio-6, Thio-7 and Thio-8. For the better inhibitors, molecular docking results suggested that the oxygen present in the para position of the aromatic ring is essential for the tyrosinase inhibition, due its high ability for complexation with Cu2+ ions. Inside the active protein pocket, Thio-2 - the best studied inhibitor - is able to interact with the amino acid residues His-155, Gly-170 and Val-172 via hydrogen bonding and hydrophobic force. Thio-2, containing a substituent on the aromatic ring similar to the substrate l-DOPA, showed a competitive inhibition mechanism as viewed in a Lineweaver-Burk plot. The same results were observed in the UV-Vis curves.

Mesoionic compounds with antifungal activity against Fusarium verticillioides.

BACKGROUND: Fungi contaminate the food of humans and animals, are a risk to health, and can cause financial losses. In this work, the antifungal activities of 16 mesoionic compounds (MI 1-16) were evaluated against mycotoxigenic fungi, including Aspergillus spp., Fusarium verticillioides and Penicillium citrinum. Furthermore, the decreased ergosterol in the total lipid content of Fusarium verticillioides was investigated. RESULTS: F. verticillioides was the most sensitive fungus to the mesoionic compounds. Among the evaluated compounds, MI-11 and MI-16 presented higher antifungal effects against F. verticillioides, with MIC values of 7.8 µg/ml, and MI-2 and MI-3 followed, with MICs of 15.6 µg/ml. The most active compounds were those with heterocyclic ring phenyl groups substituted by electron donor moieties (MI-11 and MI-16). Among some compounds with higher activity (MI-2, MI-11 and MI-16), decreased ergosterol content in the total lipid fraction of F. verticillioides was demonstrated. MI-2 reduced the ergosterol content approximately 40% and 80% at concentrations of 7.8 µg/ml and 15.6 µg/ml, respectively, and MI-11 and MI-16 decreased the content by 30% and 50%, respectively, when at a concentration of 7.8 µg/ml. CONCLUSION: These findings indicate that mesoionic compounds have significant antifungal activity against F. verticillioides.

Quantitative Structure-Property Relationship (QSPR) Models for a Local Quantum Descriptor: Investigation of the 4- and 3-Substituted-Cinnamic Acid Esterification.

In this work, the theoretical description of the 4- and 3-substituted-cinnamic acid esterification with different electron donating and electron withdrawing groups was performed at the B3LYP and M06-2X levels, as a two-step process: the O-protonation and the nucleophile attack by ethanol. In parallel, an experimental work devoted to the synthesis and characterization of the substituted-cinnamate esters has also been performed. In order to quantify the substituents effects, quantitative structure-property relationship (QSPR) models based on the atomic charges, Fukui functions and the Frontier Effective-for-Reaction Molecular Orbitals (FERMO) energies were investigated. In fact, the Fukui functions, ƒâºC and ƒ(-)O, indicated poor correlations for each individual step, and in contrast with the general literature, the O-protonation step is affected both by the FERMO energies and the O-charges of the carbonyl group. Since the process was shown to not be totally described by either charge- or frontier-orbitals, it is proposed to be frontier-charge-miscere controlled. Moreover, the observed trend for the experimental reaction yields suggests that the electron withdrawing groups favor the reaction and the same was observed for Step 2, which can thus be pointed out as the determining step.

1H NMR for quantifying sulfide trapping efficiency by using 1,3,5-tris(2-hydroxyethyl)-1,3,5-triazinane.

Hydrogen sulfide (H2S) is an extremely toxic colourless gas; it is corrosive and denser than air. It usually happens in oil and natural gas fields, refineries, coal mines, and in some industrial effluent treatment systems. This work presents an alternative method of monitoring and quantifying H2S trapping efficiency by using 1,3,5-tris(2-hydroxyethyl)-1,3,5-triazinane as a sequestering agent, and sodium sulfide as a source of sulfide ion, through (1)H NMR spectroscopy. The results proved that the reaction occurs very quickly at 20 °C at pH 7 and 10. 3,5-di(2-hydroxyethyl)-1,3,5-thiodiazinane and 5-(2-hydroxyethyl)-1,3,5-dithiozinane were observed and quantified; it was evidenced that (1)H NMR spectroscopy can be applied as a fast and effective method to quantify H2S trapping efficiency.

Evaluation of the antitumoral effects of the mesoionic compound MI-D: Implications for endothelial cells viability and angiogenesis inhibition.

Angiogenesis is considered one of the hallmarks of cancer, assisting tumor progression and metastasis. The mesoionic compound, MI-D, can induce cell death and provoke cytoskeletal and metabolic changes in cancer cells. Using in vitro and in vivo models, this study aimed to evaluate the effects of MI-D on the viability of human endothelial cells (EC) and its ability to inhibit tumor-induced angiogenesis induced by tumoral cells. For in vitro analysis, colon carcinoma (HT29) and endothelial (EA.hy926) cells were used as the tumoral and angiogenesis models, respectively. To evaluate cytotoxicity, methylene blue viability stain and annexin-V/7AAD tests were performed with both cell types. For the angiogenesis experiments, scratch wound healing and capillary tube-like formation assays were performed with the EC. The in vivo tests were performed with the chorioallantoic membrane (HET-CAM) methodology, wherein gelatin sponge implants containing MI-D (5, 25, and 50 µM), HT29 cells, or both were grafted in the CAM. Our data showed that MI-D induced apoptosis in both endothelial and colon carcinoma cells, with a strong cytotoxic effect on the tumoral lineage. The drug inhibited the EC's migration and capillary-like structure formation in vitro. In the HET-CAM assays, MI-D reduced the number of blood vessels in the membrane when grafted alone and accompanied by tumor cells. In this study, MI-D interfered in important steps of angiogenesis, such as maintenance of endothelial cell viability, migration, formation of capillary-like structures, as well tumor-induced neovascularization, reinforcing the hypothesis that MI-D might act as an inhibitor of angiogenesis, and a potential antitumor agent.

In vitro evaluation of 4-phenyl-5-(4'-X-phenyl)-1,3,4-thiadiazolium-2-phenylaminide chlorides and 3[N-4'-X-phenyl]-1,2,3-oxadiazolium-5-olate derivatives on nitric oxide synthase and arginase activities of Leishmania amazonensis.

Leishmaniasis is a spectrum of infectious diseases caused by Leishmania protozoan parasites. The purpose of this study was to perform, in vitro, a comparative analysis of the activity amastigotes. Results showed excellent efficacy of all compounds against axenic amastigotes, compared to pentamidine isethionate, the reference drug used. The cytotoxic effect of these mesoionic compounds of six mesoionic compounds (three 1,3,4-thiadiazolium-2-aminide and three 1,2,3-oxadiazolium-5-olate class compounds) was evaluated in mouse peritoneal macrophages using MTT assay, low toxicity (≈ 10%) for these mammalian cells being observed. In an attempt to define a potential drug target, the activities of nitric oxide synthase (NOS) and arginase of the parasites treated with the mesoionic derivatives were evaluated. NOS was purified from a cell-free extract of infective promastigotes and axenic amastigotes and all derivatives tested were able to inhibit the enzyme as monitored by the decrease of NADPH consumption. Arginase activity from both stages of the parasite was measured using urea production and none of the compounds inhibited the enzyme activity of axenic amastigotes. However, the compounds without substituents (MI-H and SID-H) were able to inhibit arginase activity of these parasites.

P-glycoprotein efflux pump plays an important role in Trypanosoma cruzi drug resistance.

Drug resistance in protozoan parasites has been associated with the P-glycoprotein (Pgp), an energy-dependent efflux pump that transports substances across the membrane. Interestingly, the genes TcPGP1 and TcPGP2 have been described in Trypanosoma cruzi, although the function of these genes has not been fully elucidated. The main goal of this work was to investigate Pgp efflux pump activity and expression in T. cruzi lines submitted to in vitro induced resistance to the compounds 4-N-(2-methoxy styryl)-thiosemicarbazone (2-Meotio) and benznidazole (Bz) and to verify the stability of the resistant phenotypes during the parasite life cycle. We observed that the EC50 values for the treatment of epimastigotes with 2-Meotio or Bz were increased at least 4.7-fold in resistant lines, and this phenotype was maintained in metacyclic trypomastigotes, cell-derived trypomastigotes, and intracellular amastigotes. However, in epimastigotes, 2-Meotio resistance is reversible, but Bz resistance is irreversible. When compared with the parental line, the resistant lines exhibited higher Pgp efflux activity, reversion of the resistant phenotypes in the presence of Pgp inhibitors, cross-resistance with Pgp modulators, higher basal Pgp ATPase activity, and overexpression of the genes TcPGP1 and TcPGP2. In conclusion, the resistance induced in T. cruzi by the compounds 2-Meotio and Bz is maintained during the entire parasite life cycle. Furthermore, our data suggest the participation of the Pgp efflux pump in T. cruzi drug resistance.

Novel Zn(II)-complex with hybrid chalcone-thiosemicarbazone ligand: Synthesis, characterization, and inhibitory effect on HTLV-1-infected MT-2 leukemia cells.

Chalcone and thiosemicarbazone have attracted attention due to their easy synthetic procedure and high success in the development of antiviral and antitumor, however, there are few biological data on the evaluation of chalcone-thiosemicarbazone hybrids and their complexation with metal ions. In this sense, the present work reports the synthesis and characterization of the hybrid (Z)-2-((E)-3-(4-chlorophenyl)-1-phenylallylidene)hydrazine-1-carbothioamide (CTCl) and its Zn(II)-complex (CTCl-Zn). The compounds were cell-based evaluated in terms of cytotoxicity against human T-cell lymphotropic virus type 1 (HTLV-1) infected leukemia cells (MT-2) and the experimental data were correlated with molecular docking calculations. The ligand and Zn(II)-complex were easily synthesized with a good yield - 57% and 79%, respectively. The dynamic of E/Z isomers with respect to the imine bond configuration of CTCl was evidenced by 1H NMR experiments in DMSO­d6, while the X-ray diffraction of CTCl-Zn showed that Zn(II) ion is tetracoordinated to two ligands in a bidentate mode and the metal ion lies on an intermediate geometry between the see-saw and trigonal pyramid. The ligand and complex exhibited low toxicity and the Zn(II)-complex is more cytotoxic than the ligand, with the corresponding IC50 value of 30.01 and 47.06 µM. Both compounds had a pro-apoptotic effect without the release of reactive oxygen species (ROS) and they can interact with DNA via minor grooves driven by van der Waals forces.

The in vivo activity of 1,3,4-thiadiazolium-2-aminide compounds in the treatment of cutaneous and visceral leishmaniasis.

OBJECTIVES: Researchers have recently investigated the biological activities of mesoionic (MI) compounds, which have shown in vitro activity against many species of Leishmania, as well as Trypanosoma cruzi. The main goal of this study was to evaluate and compare the activity of three MI compounds against Leishmania amazonensis and Leishmania infantum infection in vivo. METHODS: The experiments were carried out using BALB/c mice infected with L. amazonensis or L. infantum as a highly sensitive murine model. The infected mice were treated with MI-HH, MI-4-OCH(3), MI-4-NO(2) or meglumine antimoniate by different routes (intralesional, topical or intraperitoneal). RESULTS: Treatment with MI-4-OCH(3) and MI-4-NO(2) efficiently contained the progression of cutaneous and visceral leishmaniasis in comparison with the control group or mice treated with meglumine antimoniate. Interestingly, these MI compounds did not produce toxicological effects after treatment. Furthermore, treatment with these compounds led to a modulation of the immune response that was correlated with disease control. In this study, MI compounds, and MI-4-NO(2) in particular, exhibited high activity in the L. infantum murine model. In the L. amazonensis model, intralesional treatment with MI-4-OCH(3) or MI-4-NO(2) showed greater therapeutic efficacy than treatment with meglumine antimoniate, and the new topical formulations of these compounds also displayed great activity in the cutaneous leishmaniasis model. CONCLUSIONS: Upon comparison of each MI compound, MI-4-NO(2) was clearly the compound with the greatest activity in these two in vivo infection models by each administration route tested.

Effect of sydnone SYD-1 on certain functions of LPS-stimulated macrophages.

In an earlier article, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) inhibits electron transport in the respiratory chain and uncouples oxidative phosphorylation, and postulated that these effects are probably involved in its antitumor activity. We now report the effect of SYD-1 on certain macrophage functions, considering the important role of these cells in inflammatory response and also the relevant anti-inflammatory activity reported for some sydnones. Incubation of macrophages with SYD-1 (5-100 µM) for 48 h did not affect the cell viability up to a concentration of 50 µM. However, at the highest concentration (100 µM), the compound decreased macrophage viability by ~20%. In assays involving 2 h and 24 h of incubation, SYD-1 (5-100 µM) did not affect the cell viability. The incubation of macrophages with the compound for 2 h promoted a dose-dependent reduction of phagocytic activity of up to ~65% (100 µM). SYD-1 (100 µM) was also able to increase the production of superoxide anion (~50%). In the absence of LPS, SYD-1 decreased NO production dose-dependently by up to ~80% (100 µM). When SYD-1 and LPS were incubated concomitantly, the decrease of NO promoted by SYD was the most pronounced, reaching up to ~98% at the same concentration (50 µM). SYD-1 dose-dependently suppressed IL-6 secretion by LPS-stimulated macrophages, reaching up to ~90% of inhibition at the highest concentration (100 µM). These results indicate that SYD-1 promotes effects similar to those described for anti-inflammatory and immunosuppressive drugs, thus motivating further studies to clarify the mechanisms involved in this activity.

Investigation of trypanothione reductase inhibitory activity by 1,3,4-thiadiazolium-2-aminide derivatives and molecular docking studies.

The biological activities of a series of mesoionic 1,3,4-thiadiazolium-2-aminide derivatives have been studied. The most active compounds (MI-HH; MI-3-OCH(3); MI-4-OCH(3) and MI-4-NO(2)) were evaluated to determine their effect on trypanothione reductase (TryR) activity in Leishmania sp. and Trypanosoma cruzi. Among the assayed compounds, only MI-4-NO(2) showed enzyme inhibition effect on extracts from different cultures of parasites, which was confirmed using the recombinant enzyme from T. cruzi (TcTryR) and Leishmania infantum (LiTryR). The enzyme kinetics determined with LiTryR demonstrated a non-competitive inhibition profile of MI-4-NO(2). A molecular docking study showed that the mesoionic compounds could effectively dock into the substrate binding site together with the substrate molecule. The mesoionic compounds were also effective ligands of the NADPH and FAD binding sites and the NADPH binding site was predicted as the best of all three binding sites. Based on the theoretical results, an explanation at the molecular level is proposed for the MI-4-NO(2) enzyme inhibition effect. Given TryR as a molecular target, it is important to continue the study of mesoionic compounds as part of a drug discovery campaign against Leishmaniasis or Chagas' disease.

Solvent-free synthesis, DNA-topoisomerase II activity and molecular docking study of new asymmetrically N,N'-substituted ureas.

A new series of asymmetrically N,N'-substituted ureas 20–25 was prepared using solvent free conditions, which is an eco-friendly methodology, starting with Schiff bases derived from cinnamaldehyde and p-substituted anilines, which are subsequently submitted to reduction reactions that afford the corresponding asymmetric secondary amines. All of the intermediates were prepared using solvent free reactions, which were compared to traditional methodologies. All of the reactions required a remarkably short amount of time and provided good yields when solvent free conditions were employed compared to other methodologies. The DNA-topoisomerase II-α (topo II-α) activity was evaluated in relaxation assays, which showed that all of the compounds inhibited the enzyme activity at 10 μM, except for urea 24. Furthermore, a molecular docking study indicated that the compounds 20–25 binding to the topo II-α are able to interact with the same binding site as the anticancer drug etoposide, suggesting that the ureas could inhibit the enzyme by the same mechanism of action observed for etoposide, which prevents re-ligation of the DNA strands.

Evaluation of thiosemicarbazones and semicarbazones as potential agents anti-Trypanosoma cruzi.

Synthetic thiosemicarbazones and semicarbazones were evaluated for their Trypanosoma cruzi trypomastigotes obtained from LLC-MK2 cell cultures. In general, thiosemicarbazone derivatives were most effective and among them the 4-N-(2'-methoxy styryl)-thiosemicarbazone was chosen, to compare the in vitro effect against amastigotes of T. cruzi lodged in mouse peritoneal and human macrophages. A potent trypanocidal effect was observed that was more pronounced against parasites internalized in human macrophages. A potential target for this compound was also evaluated by measuring the nitric oxide synthase activity through NADPH consumption. A significant decrease in enzyme activity was observed. In contrast to the cytotoxic effect observed with benznidazole, no macrophage toxicity was observed for any of the compounds, indicating that their activity was specific for the parasite forms investigated.

Microwave-assisted synthesis of new N1,N4-substituted thiosemicarbazones.

We present an efficient procedure for the synthesis of thirty-six N1,N4-substituted thiosemicarbazones, including twenty-five ones that are reported for the first time, using a microwave-assisted methodology for the reaction of thiosemicarbazide intermediates with aldehydes in the presence of glacial acetic acid in ethanol and under solvent free conditions. Overall reaction times (20-40 min when ethanol as solvent, and 3 min under solvent free conditions) were much shorter than with the traditional procedure (480 min); satisfactory yields and high-purity compounds were obtained. The thiosemicarbazide intermediates were obtained from alkyl or aryl isothiocyanates and hydrazine hydrate or phenyl hydrazine by stirring at room temperature for 60 min or by microwave irradiation for 30 min, with lower yields for the latter. The preliminary in vitro antifungal activity of thiosemicarbazones was evaluated against Aspergillus parasiticus and Candida albicans.

The toxicity of 1,3,4-thiadiazolium mesoionic derivatives on hepatocarcinoma cells (HepG2) is associated with mitochondrial dysfunction.

Mesoionic compounds, 4-phenyl-5-(4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X = OH; MI-D: X = NO2), possess significant antitumor and cytotoxic effects on several cancer cells. In this work, we evaluated the cytotoxicity of MI-J and MI-D on human hepatocellular carcinoma (HepG2 cells) grown in either high glucose (HG) or galactose medium (GAL) to clarify whether the effects of mesoionics on mitochondrial bioenergetics are associated with their cytotoxicity in these cells. MI-J and MI-D (5-50 µM) decreased the viability of HepG2 cells in a dose- and time-dependent manner, as assessed by MTT, LDH release and dye with crystal violet assays. Both compounds at lower (5 µM) and intermediate (25 µM) concentrations were more toxic to cells grown in GAL medium. MI-J inhibited the basal state of respiration in HepG2 cells cultured in HG and GAL media; however, in GAL medium, this effect occurred at the lowest concentration (5 µM). A leak-state stimulus was observed only after incubation with MI-J (5 µM) for GAL medium. MI-D stimulated and inhibited the leak state in cells grown in HG medium at concentrations of 5 µM and 25 µM, respectively. In cells cultured in GAL medium, respiration was strongly inhibited by MI-D at the highest concentration (25 µM). In contrast, at 5 µM, the mesoionic inhibited the basal and uncoupled states at 30% and 50%, respectively. The inhibition of the basal state by MI-J and MI-D was consistent with the increase in lactate levels in both media, which was higher for the GAL medium. Both mesoionics slightly decreased pyruvate levels only in cells cultured in GAL medium. Additionally, MI-J (25 µM) reduced the ATP amount in cells cultured in both media, while MI-D (25 µM) promoted a reduction only in cells grown in GAL medium. Our results show that MI-J and MI-D depress mitochondrial respiration and consequently change metabolism and reduce ATP levels, effects associated with their toxicity in hepatocarcinoma cells.