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.     

Thiosemicarbazones derived from 1-indanones as new anti-Trypanosoma cruzi agents

In the present work, we synthesized a series of thiosemicarbazones derived from 1-indanones with good anti-Trypanosoma cruzi activity. Most of them displayed remarkable trypanosomicidal activity. All the compounds showed nonspecific cytotoxicity on human erythrocytes. The ability of the new compounds to inhibit cruzipain, the major cysteine protease of T. cruzi, was also explored. Thiosemicarbazones 12 and 24 inhibited this enzyme at the dose assayed. This interaction was also studied in terms of molecular docking.     

Platinum(II) metal complexes as potential anti-Trypanosoma cruzi agents

In the search for new therapeutic tools against Chagas’ disease (American Trypanosomiasis) two series of new platinum(II) complexes with bioactive 5-nitrofuryl containing thiosemicarbazones as ligands were synthesized, characterized and in vitro evaluated. Most of the complexes showed IC₅₀ values in the μM range against two different strains of Trypanosoma cruzi, causative agent of the disease, being as active as the anti-trypanosomal drug Nifurtimox. In particular, the coordination of L3 (4-ethyl-1-(5-nitrofurfurylidene)thiosemicarbazide) to Pt(II) forming [Pt(L3)₂] lead to almost a five-fold activity increase in respect to the free ligand. Trying to get an insight into the trypanocidal mechanism of action of these compounds, DNA and redox metabolism (intra-parasite free radical production) were evaluated as potential parasite targets. Results suggest that the complexes could inhibit parasite growth through a dual mechanism of action involving production of toxic free radicals by bioreduction and DNA interaction.     

Imidazolidines as new anti-Trypanosoma cruzi agents: biological evaluation and structure-activity relationships

Imidazolidine derivatives were studied as anti-Trypanosoma cruzi agents. Imidazolines can be considered as ethylenediamine/carbonyl precursors and therefore interfere with the biosynthesis of polyamines into the parasite. Some of the derivatives were found to have high and selective activity against the proliferative stages of the parasite, with IC(50) values against the epimastigote form in the low micromolar range as the reference drug Nifurtimox. The imidazolidines demonstrated to be stable after five days of incubation in buffer glucose, pH 7, indicating that diamines were not obtained in these conditions. But it was found that two of the studied diamine precursors were as active as the parent compounds. Probably, the imidazolidines affect the mitochondrial integrity according to the excreted end-products found in the NMR studies. The QSAR studies indicated that the bioactivities are correlated with the lipophilicities. In conclusion, we have described a new and relevant bioactivity for imidazolidines. The results support further in vivo studies of some of these imidazolidine derivatives.     

Heteroallyl-containing 5-nitrofuranes as new anti-Trypanosoma cruzi agents with a dual mechanism of action

New heteroallyl-containing 5-nitrofuranes were synthesized as potential anti-Trypanosoma cruzi agents with a dual mechanism of action, oxidative stress and inhibition of membrane sterol biosynthesis. Some of the derivatives were found to have high and selective activity against the proliferative stages of the parasite, with IC(50) values against the clinically relevant intracellular amastigote forms in the low micromolar to sub-micromolar range. Oxidative stress was verified measuring cyanide dependent respiration. Inhibition of the de novo sterol biosynthesis at the level of squalene epoxidase was confirmed, using high-resolution gas-liquid chromatography coupled to mass spectrometry, by the disappearance of the parasite's mature sterols and the concomitant accumulation of squalene. The in vitro activities of these novel compounds were superior to that of nifurtimox, a nitrofuran currently used in the treatment of human Chagas' disease, and terbinafine, a commercially available allylamine-based squalene epoxidase inhibitor. The results support further in vivo studies of some of these nitrofuran derivatives.     

Mutagenicity of CL 64855, a potent anti-Trypanosoma cruzi drug

The nitroimidazole-thiadiazole derivative CL 64855 (2-amino-5 (1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole), a potent antimicrobial agent with curative action against Trypanosoma cruzi, was assayed in the Salmonella/microsome test. CL 64855 proved to be a potent mutagen to the frameshift indicator tester strains TA98 and TA102. No activity was observed with the base-pair substitution mutagen indicator strain TA100 in spot tests. No significant increase in the number of induced mutants could be detected in the presence of rat-liver microsome fraction. The excision-repair-deficient strain TA98 was much more sensitive to the killing action of CL 64855 than TA102, a repair-proficient strain. Possible differences among the mutagenic effects of CL 64855 and those observed with other anti-trypanosomal drugs are discussed.     

New potent imidazoisoquinolinone derivatives as anti-Trypanosoma cruzi agents: Biological evaluation and structure–activity relationships

A series of novel benzoimidazo and N-aryl-5-oxo-imidazo[1,2-b]isoquinoline-10-carbothioamides was developed. All the compounds were evaluated for their in vitro action against the epimastigote form of Trypanosoma cruzi. Four of them showed higher activity than Nifurtimox. Their unspecific cytotoxicity was evaluated using HeLa and L6 cells, being non-toxic at concentrations at least 15 and 200 times higher than that of T. cruzi IC_50. To gain insight into the mechanism of action, their DNA binding properties and reactivity with glutathione were studied, and QSAR study was performed.     

In vitro phenotypic screening of 7-chloro-4-amino(oxy)quinoline derivatives as putative anti-Trypanosoma cruzi agents

In this study, a series of 22 pre-synthesized 7-chloro-4-amino(oxy)quinoline derivatives was assayed in vitro as potential antichagasic agents. A primary screening against Trypanosoma cruzi epimastigotes and a non-specific cytotoxicity assay on murine fibroblasts were simultaneously performed, resulting quinolines 3, 7 and 12 with great selectivity (SI) on the extracellular parasite (SI₇, SI₃, SI₁₂ and SI_BZ >9.44). Therefore, the activity of these derivatives was evaluated on intracellular amastigotes, achieving derivative 7 the best SI (SI = 12.73). These results, supported by the in silico prediction of a good oral bioavailability and a suitable risk profile, propose the 4-amino-7-chloroquinoline scaffold as a potential template for designing trypanocidal prototypes.     

CL 64,855, a potent anti-Trypanosoma cruzi drug, is also mutagenic in the Salmonella/microsome assay

The nitroimidazole-tiadiazole derivative CL 64,855 (2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole, a potent anti-trypanosomal drug, was assayed in a short-term bacterial mutagenicity test with Salmonella typhimurium strains TA 98, TA 100 and TA 102. Results indicate that CL 64,855 is a potent frameshift mutagen detected by strains TA 98 and TA 102. CL 64,855 was able to revert the indicators strains at concentrations as low as 0.1 micrograms/plate. Metabolic activation experiments with rat liver microsomal fractions did not increase the mutagenic action of CL 64,855.     

5-Nitro-2-furfuriliden derivatives as potential anti-Trypanosoma cruzi agents: Design,synthesis, bioactivity evaluation,cytotoxicity and exploratory data analysis

The anti-Trypanosoma cruzi activity of 5-nitro-2-furfuriliden derivatives as well as the cytotoxicity of these compounds on J774 macrophages cell line and FN1 human fibroblast cells were investigated in this study. The most active compounds of series I and II were 4-butyl-[N′-(5-nitrofuran-2-yl) methylene] benzidrazide (3g; IC₅₀ = 1.05 μM ± 0.07) and 3-acetyl-5-(4-butylphenyl)-2-(5-nitrofuran-2-yl)-2,3-dihydro,1,3,4-oxadiazole (4g; IC₅₀ = 8.27 μM ± 0.42), respectively. Also, compound 3g was more active than the standard drugs, benznidazole (IC₅₀ = 22.69 μM ± 1.96) and nifurtimox (IC₅₀ = 3.78 μM ± 0.10). Regarding the cytotoxicity assay, the 3g compound presented IC₅₀ value of 28.05 μM (SI = 26.71) against J774 cells. For the FN1 fibroblast assay, 3g showed IC₅₀ value of 98 μM (SI = 93.33). On the other hand, compound 4g presented a cytotoxicity value on J774 cells higher than 400 μM (SI >48), and for the FN1 cells its IC₅₀ value was 186 μM (SI = 22.49). Moreover, an exploratory data analysis, which comprises hierarchical cluster (HCA) and principal component analysis (PCA), was carried out and the findings were complementary. The molecular properties that most influenced the compounds’ grouping were C log P and total dipole moment, pointing out the need of a lipophilic/hydrophilic balance in the designing of novel potential anti-T. cruzi molecules.     

DNA as molecular target of analogous palladium and platinum anti-Trypanosoma cruzi compounds: a comparative study

In the search for drugs with anti-trypanosome activity, we had previously synthesized two series of platinum and palladium analogous compounds of the formula [M^IICl₂(HL)], where HL were bioactive 5-nitrofuryl or 5-nitroacroleine thiosemicarbazone derivatives. In this work, we thoroughly characterized [M^IICl₂(HL)] complexes interaction with DNA by using different techniques: gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM). Electrophoresis results showed that all complexes induced a withdrawal of DNA superhelicity demonstrated by a decrease in electrophoretic mobility of supercoiled DNA form. This effect on migration was dependent on dose but also on the nature of both the metal and the ligand. In general, the effect produced by palladium complexes was significantly more intense than that observed for the corresponding platinum analogs. Differences between palladium and platinum complexes were also observed in CD experiments. While palladium complexes induce evident calf thymus (CT)-DNA profile changes compatible with B-DNA to Z-DNA conformational transition, no clear effect was observed for platinum ones. Additionally, AFM studies showed that changes in the shape of plasmid DNA, like supercoiling, kinks and thickness increase resulted more intense for the former. In addition, either Pd or Pt complexes increased the viscosity of CT DNA solutions in a concentration dependent manner. Although the nature of DNA interaction of both series of analogous palladium and platinum complexes seemed to be similar, an explanation for the observed differential intensity of the effect could be related to the known kinetic stability differences between palladium and platinum compounds.     

Novel l-adenosine analogs as cardioprotective agents

Two l-nucleosides, l-3′-amino-3′-deoxy-N⁶-dimethyladenosine (l-3′-ADMdA) 1, previously synthesized in our laboratory, and the novel l-3′-amino-3′-deoxy-N⁶-methyladenosine-5′-N-methyluronamide (l-3′-AM-MECA) 2 were evaluated in an ischemia/reperfusion model on Langendorff perfused mouse heart. l-3′-ADMdA 1 was found to enhance functional recovery from ischemia (32.2 ± 3.7 cm H₂O/s % rate pressure product, compared to 21.3 ± 1.4 for the control and 30.7 ± 3.4 for adenosine) and increase the time to onset of ischemic contracture (14.5 ± 0.9 min, compared to 10.5 ± 1.0 min for the control and 13.6 ± 0.6 min for adenosine) comparable to adenosine. Consistent with the functional recovery data, decreased infarction area was seen in the case of 1 (19.1 ± 8.4, compared to 40.5 ± 7.2% for the control and 11.5 ± 2.1% for adenosine). In contrast, l-3′-AM-MECA 2 did not show significant functional recovery, increased onset of contracture, nor decreased infarction area compared to control. Unlike adenosine, neither 1 nor 2 induced cardiac standstill in mouse heart.     

Synthesis and anti-Trypanosoma cruzi activity of derivatives from nor-lapachones and lapachones

New naphthoquinone derivatives were synthesized and assayed against bloodstream trypomastigote forms of Trypanosoma cruzi, the etiological agent of Chagas’ disease. The compounds were rationalized based on hybrid drugs and appear as important compounds against this parasite. From nor-lapachol were prepared five substituted ortho-naphthofuranquinones, a non-substituted para-naphthofuranquinone, a new oxyrane and an azide and from -lapachone a new non-substituted para-naphthofuranquinone. Other five substituted ortho-naphthofuranquinones recently designed as cytotoxic, were also evaluated. The most active compounds were the ortho naphthofuranquinones 3-(4-methoxyphenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione and 3-(3-nitrophenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione with trypanocidal activity higher than that of benznidazole, the standard drug. The compounds were rationalized based on hybrid drugs and appear as important compounds against T. cruzi. The trypanocidal activity of these substances endowed with redox properties representing a good starting point for a medicinal chemistry program aiming the chemotherapy of Chagas’ disease.     

Isotype determination of anti-Trypanosoma cruzi antibody in murine Chagas' disease.

Isotypic analysis of anti-parasite humoral responses of C57B1/6 and C3H (He) mice surviving acute Trypanosoma cruzi infection showed that both mouse strains demonstrate IgG1, IgG2a, IgG2b, and IgM enzyme-linked immunosorbent assay titers from days 21 to 300 of infection. Using the western blot technique to determine the antigen specificity of the isotypic responses, 100-day infected C3H mice showed strong IgG1, IgG2a, and IgG2b responses to many antigens, whereas C57B1/6 mice showed weak responses to fewer antigens. Isotype western blots showed that reactivity to the T. cruzi antigen of 75-77 kDa is present in the humoral response of day 21-infected mice that will survive and missing in those that will not survive. In general, surviving immunized C3H mice respond with IgG1, IgG2a, and IgG2b reactions to the 75-77-kDa and other antigens, whereas resistant B6 mice concentrate their anti-T. cruzi response in the IgG2b isotype to the 75-77-kDa antigen. Perhaps induction of ineffective antibody responses to nonprotective antigens is beneficial to the parasite and detrimental to the host.     

Structure-based design of UDP-GlcNAc analogs as candidate GnT-V inhibitors

Background: N-Glycan branching regulates various functions of glycoproteins. N-Acetylglucosaminyltransferase V (GnT-V) is a GlcNAc transferase that acts on N-glycans and the GnT-V-producing branch is highly related to cancer progression. This indicates that specific GnT-V inhibitors may be drug candidates for cancer treatment. To design novel GnT-V inhibitors, we focused on the unique and weak recognition of the donor substrate UDP-GlcNAc by GnT-V. On the basis of the catalytic pocket structure, we hypothesized that UDP-GlcNAc analogs with increasing hydrophobicity may be GnT-V inhibitors.Methods: We chemically synthesized 10 UDP-GlcNAc analogs in which one or two phosphate groups were replaced with hydrophobic groups. To test these compounds, we set up an HPLC-based enzyme assay system for all N-glycan-branching GlcNAc transferases in which GnT-I–V activity was measured using purified truncated enzymes. Using this system, we assessed the inhibitory effects of the synthesized compounds on GnT-V and their specificity.Results: Several UDP-GlcNAc analogs inhibited GnT-V activity, although the inhibition potency was modest. Compared with other GnTs, these compounds showed a preference for GnT-V, which suggested that GnT-V was relatively tolerant of hydrophobicity in the donor substrate. Docking models of the inhibitory compounds with GnT-V suggested the mechanisms of how these compounds interacted with GnT-V and inhibited its action.Conclusions: Chemical modification of the donor substrate may be a promising strategy to develop selective inhibitors of GnT-V.General significance: Our findings provide new insights into the design of GnT inhibitors and how GnTs recognize the donor substrate.     

Structure-activity studies of quinuclidinone analogs as anti-proliferative agents in lung cancer cell lines

We have synthesized and tested novel quinuclidinone analogs to assay the effects on H1299 lung cancer cell lines alone or with gamma-radiation. We have found two series of quinuclidinone analogs that act as anti-cancer agents. Of these, four interesting analogs significantly decreased cell viability in H1299 lung cancer cell lines. Two derivatives decreased cell proliferation in a dose-dependent fashion alone or in the presence of gamma-radiation. Radiosensitization increased when derivative treatment preceded radiation treatment for both derivatives. These preliminary studies show an evidence for both additive and synergistic cytotoxicity for treatment of lung cancer by these novel quinuclidinone analogs.     

In vitro and in vivo anti-Trypanosoma cruzi activity of a novel nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.