PL237553B1 - Medical use of 4-(2-fluorophenyl)-1-(4-methylimidazol-5-yl)thiosemicarbazide - Google Patents

Abstract

The subject of the application is 4-(2-fluorophenyl)-1-(4-methylimidazol-5-yl)thiosemicarbazide, for use in the treatment of toxoplasmosis. Furthermore, the application also covers the use of 4-(2-fluorophenyl)-1-(4-methylimidazol-5-yl)thiosemicarbazide for the manufacture of a medicament intended for the treatment of toxoplasmosis.

Description

Description of the invention

The subject of the invention is the medical use of 4- (2-fluorophenyl) -1- (4-methylimidazol-5-ylcarbonaylo) thiosemicarbazide of the general formula shown in the figure, especially for the treatment of toxoplasmosis.

Derivatives of 1,4-disubstituted thiosemicarbazide and their cyclic thiadiazole and triazole analogues are known from the literature (Eur J Med Chem 2010, 45, 3685; Antimicrob Agents Chemother 2014, 58, 7583; Molecules 2014, 19, 9926), which show antiparasitic activity against the pathogenic parasite Toxoplasma gondii. Their biological activity is comparable or better than that of the commonly used sulfadiazine drug. Unfortunately, some of them turned out to be toxic. On the other hand, Walchshofer et al. (Bioorg Med Chem 2000, 10, 871) described a series of fused furan-isoquinoline derivatives which, at a concentration of 2 pg / mL, showed inhibitory activity against T. gondii in the percentage range from 56 to 96.5. Also the fluoroquinolone derivative showed significant biological activity, comparable to the drug trovafloxacin; unfortunately, its toxic effect has not been assessed for it (Bioorg Med Chem Lett 2004, 14, 2773). A series of thiazolidinone and thiosemicarbazone derivatives has been reported by Goes et al. (Bioogr Med Chem Lett 2005, 15, 2575). Unfortunately, among the tested compounds, only selected derivatives showed a biological activity higher than that of sulfadiazine or hydroxycarbamide.

The aim of the invention was to determine thiosemicarbazide derivatives which would show high antiparasitic activity against the pathogenic parasite Toxoplasma gonidii, which would exceed that of the known compounds of this group.

Toxoplasma gondii is a cosmopolitan parasite that causes toxoplasmosis in all mammalian species, including humans and birds. According to the World Health Organization, it is estimated that even 1/3 of the human population has been invaded by this parasite. In Poland, infection occurs in 50-60% of people (Int J Parasitol 2000, 30, 1217; Mikrobiol Med 1996, 1, 14; Ann Agric Environ Med 2001, 8, 25). However, a significant number of infections are not recorded, because toxoplasmosis in healthy people is usually asymptomatic, in most cases in a way that does not require any therapy or medical intervention. The most common source of infection is food: raw or undercooked meat, vegetables, fruit and water (horizontal route) (Clin Infect Dis 2007, 45, 88; Adv Food Nutr Res 2010, 60, 1; Epidemiol Infect 1999, 122, 305). Another way of infection with the so-called vertical disease occurs in pregnant women, as a result of which protozoa may be transferred from the mother to the developing fetus (congenital toxoplasmosis) (Parasitology 2011,9, 1; J Pediatr Health Care 2011,25, 355).

Depending on the form of the disease, different treatment regimens are used. Different centers also recommend different treatment times. Most often, combination therapy is used or individual drugs are administered consecutively. Combined therapy consists in administering pyrimethamine in combination with sulfadiazine with simultaneous supplementation with folinic acid (Toxoplasmosis. Med 2005, 33, 120; Drugs 1997, 53, 40; Antimicrob Agents Chemother 2008, 52, 1269; Drug Discovery Today 2005, 10, 121 ).

The exception is infected pregnant women under 6 months of pregnancy who are administered spiromycin, which has high affinity for the placenta, but unfortunately has a low protective effect (Zdrowie Publiczne 2010, 120, 80). In the treatment of ocular toxoplasmosis, in addition to the standard combination of pyrimethamine with sulfonamides, trimethoprim with sulfamethoxazole, atovaquone, azithromycin and anti-inflammatory steroid drugs are also used (Int J Med Sci 2009, 6, 140).

Unfortunately, traditional treatment of toxoplasmosis has numerous side effects. During treatment, both haematological symptoms (megaloblastic anemia, thrombocytopenia, leukopenia) and neurological disorders (headache, dizziness, ataxia, seizures, insomnia and depression), as well as changes in the skin and mucous membranes (dermatitis, abnormal pigmentation, rash, glossitis). It may also occur: dry mouth, gastrointestinal disorders, fever, bone marrow damage, teratogenicity, nephrolithiasis, resistance (Int J Med Sci 2009, 6, 140; Wiad Parazytol 2011, 57, 87; Mutat Mes 1998, 415, 69; Toxicol Lett 1982, 10, 51; Antimicrob Agents Chemother 2008, 52, 1269; Drugs 2004, 64, 245; Ann Allergy Asthma Immunol 2008, 100, 91).

As is clear from the above facts, the use of classic drugs for the treatment of toxoplasmosis is associated with significant risks. Moreover, due to the narrow therapeutic range of pyrimethamine (0.08-0.6 µg / mL blood), it is difficult to determine the correct dose of the drug. As reported by Lipka et al: (Wiad Parazytol 2011, 57, 87), only 58.3% of treated children had adequate serum pyrimethamine levels, while 29.2% showed a value lower than the suggested therapeutic dose, and 12.5%

PL 237 553 Β1 of the respondents exceeded this norm. There is therefore a real need to develop new drugs capable of effectively treating toxoplasmosis without the side effects discussed above.

The 4- (2-fluorophenyl) -1- (4-methylimidazol-5-yl) thiosemicarbazide of the invention for use in the treatment of toxoplasmosis.

As shown by the tests, 4- (2-fluorophenyl) -1- (4-methylimidazol-5-yl) thiosemicarbazide of the formula shown in the figure shows activity useful for the treatment of toxoplasmosis. This compound shows strong inhibition of T. gondii proliferation in vitro and no significant cytotoxicity to the host cells. Thus, this compound may find use in the preparation of new drugs for the treatment of toxoplasmosis.

A description of the tests and the results of the activity of the compound of use according to the invention are provided below.

Assessment of the effect of 4- (2-fluorophenyl) -1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide on the viability of L929 cells - MTT test

Principle of the MTT test

The L929 cell viability assay was performed using the MTT assay according to European Standard: ISO 10993-5: 2009 (E), Biological evaluation of medical devices, Part 5: Tests forin vitro cytotoxicity. The MTT assay is based on the conversion of the yellow MTT salt (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) to a violet-blue, insoluble formazan. NADPH or NADH are responsible for this conversion, produced by the enzyme mitochondrial dehydrogenase, present in metabolically active cells. The concentration of the blue product is directly proportional to the number of viable cells in the sample and can be measured spectrophotometrically.

Experimental part

On the 96 well plate (Nunc ^TC) applied to L929 cells - Catalog No. ATCC- CCL-1 ™ having a density of 1x10 ^4/100 mL / well, suspended in IMDM culture medium (Iscove's Modified Dulbecco's Medium - Cytogen) and incubated (37 ° C, 10% CO2) for 24 h. After incubation, the medium was removed from the cells and 100 μί of appropriate dilutions of the test compound (0.0; 3.91; 7.81; 15.63; 31.25; 62.50; 125.00) were added and, for comparison, sulfadiazine (Sigma), the final concentrations of which were: 0.0; 5.0; 25.0; 50.0; 125.0; 500.0; 1250.0; 2500.0 μg / mL. The effect of the solvent on cell viability was also assessed, at the final concentration in the microculture: 0.05; 0.10; 0.25; 0.5; 1; 2% DMSO. Controls were cells grown in complete IMDM culture medium.

After adding the above-mentioned of compounds, cells were incubated for 24h at 37 ° C, 10% CO 2. After this time, the cultures were observed under a microscope and pictures were taken. The media was then removed from the monolayer, and 50 μί MTT (C = 1 mg / mL, Sigma) was added to each well of the plate and incubated (37 ° C and 5% CO2). After 2 h, the plates were centrifuged (200 x g, 10 min) and the formazan crystals were dissolved by adding 150 μί DMSO / well. The color of the product was stabilized by adding 25 μί of glycine buffer. The absorbance level was measured with an ELISA reader at a wavelength of λ = 550 nm.

The significance level p was determined by the non-parametric Mann-Whitney U test using the SigmaStat program. The results with a significance level of p <0.05 were considered statistically significant. The values of CC30 (cytotoxic concentration 30 - concentration causing a cytotoxic effect for 30% of the tested cells) were determined on the basis of the analysis of the relationship between the concentrations of the tested compound and the cell survival rate (Statistica 10 PL).

Results

The experiment evaluated the effect of the test compound on the viability of the L929 host cells by means of the MTT assay. Cell cultures incubated in IMDM medium without addition of the test compound and solvent (DMSO) were used as controls. Cell viability was calculated by the formula:

The value of the absorbance of the test sample

Service life = -------------—----------— x 100%

The absorbance value of the control sample

T. gondii belongs to the obligatory intracellular parasites and its multiplication takes place only in living host cells. Therefore, only those concentrations which yield> 70% viability of the L929 lineage were selected to study the effect of the compound on the intracellular proliferation of the protozoan.

PL 237 553 Β1

According to the ISO 109935: 2009 (E) standard, a given compound can be considered non-toxic to cells if it does not decrease cell viability below 70%, therefore the CC30 value was determined by reading it from the cell survival curve [%] on the compound concentrations used and sulfadiazine [μg / mL], derived from the data presented in Table 1 and Table 2, respectively.

Due to the need to dissolve the compound in DMSO, in the first stage of the experiment, the influence of the applied solvent concentrations on the viability of the L929 cells was investigated. It was found that the concentrations of DMSO used in the experiment had no significant effect on the viability and morphology of L929 cells (Table 1).

Table 1. Cell viability [%] of the L929 line at individual DMSO concentrations ± standard deviation

DMSO concentration [%] 2.0 1.0 0.5 0.25 0.10 0.05 living cells 69.23 * 82.26 89.89 90.36 92.56 95.67 [%] ± 4.34 ± 10.02 ± 9.25 ± 5.99 ± 8.02 ± 4.59

* ^> <0.05; cell viability below 70%

Parallel to the MTT assay, microscopic observation of the microcultures incubated with different concentrations of DMSO was performed. Observation of cells under a microscope confirmed the absence of cytopathic changes in cultures with DMSO concentration up to 1%. The effect of a drug commonly used in the treatment of toxoplasmosis, sulfadiazine, on the viability of L929 cells was also assessed. The CC30 dose for sulfadiazine was found to be> 2500 μg / mL (Table 2). During microscopic analysis, no changes in the morphology of cells incubated with various concentrations of a commercially available drug were observed.

Table 2. Cell survival [%] of the L929 line at individual sulfadiazine concentrations ± standard deviation

The concentration of sulfadiazine [pg / mL] 2500.0 1250.0 500.0 125.0 50.0 25.0 5.0 CC30 [pg / mL] live cells [%] 81.01 ± 5.46 83.28 ± 2.56 94.59 ± 3.59 95.34 ± 4.79 94.59 ± 4.56 91.99 ± 3.21 81.29 ± 4.13 > 2500

The effect of the compound on the viability of L929 cells was investigated. CC30 concentrations were determined for the L929 cell line (Table 3) and pictures of the cell culture were taken with the addition of the determined concentration of the test compound.

Table 3. Cell survival [%] of the L929 line in the concentration range of 4- (2-fluorophenyl) -1- (4-methylimidazol-5-yl) thiosemicarbazide 1-125 pg / mL ± standard deviation

relationship 125.00 Compound concentration [pg / mL] 62.50 31.25 15.63 7.81 3.91 CC30 [pg / mL] 1 77.69 ± 4.56 79.75 84.33 93.60 ± 3.61 ± 3.59 ± 3.56 91.07 ± 3.48 93.13 ± 5.04 > 125

Based on the conducted studies, it was found that 4- (2-fluorophenyl) -1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide did not exhibit cytotoxicity in the concentration range of 0.0-125 μg / mL. The test compound above the concentration of 125 μg / mL precipitates in the form of crystals during the course of the experiment, which makes it impossible to accurately determine the CC30 value.

Study of the effect of 4- (2-fluorophenyl) -1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide on the proliferation of T. gondii in L929 cells (tritiated uracil incorporation test).

PL 237 553 Β1

The effect of 4- (2-fluorophenyl) -1- (4-methylimidazol-5-yl) thiosemicarbazide on the intracellular proliferation of the protozoan T. gondii strain BK in L929 cells was tested with the tritium-labeled uracil incorporation assay. The control of the experiment was mouse fibroblast cells infected with protozoa and incubated in culture medium without drug (measured value of proliferation was taken as 100%). The IC30 value (inhibitory concentration 50) was determined.

Principle of reaction

^{The 3} H-uracil incorporation test measures the level of radioactivity of the element - tritium ( ³ H), which, together with uracil, is captured and incorporated into the T. gondii RNA chain. The smaller number of counts by the scintillation reader compared to the control indicates the inhibition of protozoan multiplication in the microculture.

Experimental part

24-hour culture of L929 cells (1 x 10 ^4/50 μί / well) were infected with T. gondii strain tachyzoitami AB in the ratio 1 cell host 10 protozoan cells (1 x 10 ^5/50 μ L / well). Microcultures were incubated for 2h at 37 ° C, 10% CO2, allowing the protozoan to enter cells and form a parasitic vacuole. After this time, 100 μί were added: i) test compound to give final concentrations: 0.0; 3.91; 7.81; 15.63; 31.25; 62.50; 125.0 μg / mL, and for parallel microculture. ii) DMSO (0.0; 0.10; 0.5; 1.0; 2.0) and iii) sulfadiazine: 0.0; 5.0; 25.0; 50.0; 125.0; 500.0; 1250.0; 2500.0 μg / mL. Plates were incubated 48 h (37 ° C, 10% CO 2), then ³ H-uracil (1 μθί / 50 μί) was added to each well and incubation continued for 24 h under the conditions as above. After this time, the plates were frozen at -20 ° C. Prior to reading the assay, the plate was thawed and the contents of the wells collected on paper (PrintedFiltermat A) which was allowed to dry. A scintillation reader (WALLAC) was used to measure the level of β radiation, previously soaking the papers with scintillation fluid (BetaplateScint, PerkinElmer).

The significance level p was determined by the non-parametric Mann-Whitney U test using the SigmaStat program. The results with a significance level of p <0.05 were considered statistically significant. IC50 values were determined on the basis of the analysis of the relationship between the applied concentrations of the test compound and the degree of intracellular multiplication of the parasite (ED50plus v 1.0).

Results

In the first stage of the experiment, the effect of the solvent on the proliferation of T. gondii in L929 cells was examined (Table 4).

Table 4. Intensity of T. gondii proliferation in L929 cells cultured in the presence of various concentrations of DMSO ± standard deviation

DMSO concentration [%] 2 1 0.5 0.25 0.10 0.5 protozoan proliferation 66.32 * 70.31 99.59 101.23 99.69 99.32 [%] ± 9.52 ± 8.56 ± 9.36 ± 5.89 ± 8.68 ± 7.23

* p <0.05

The decrease in protozoan multiplication was observed only in the concentration of 2% DMSO. The positive control of the experiments on the effect of the test compound was microculture with sulfadiazine, a drug that has long been used in the treatment of toxoplasmosis (Table 5). Table 5 presents the data on its activity against T. gondii to compare the activity of the compound according to the invention with the known and used drug, while Table 6 shows the data on the activity of the compounds according to the invention. As can be seen from the data, the compound according to the invention shows higher efficacy than the known drug.

PL 237 553 Β1

Table 5. The proliferation intensity [%] of T. gondii in L929 cells cultured in the presence of sulfadiazine - a commercial drug used in the treatment of toxoplasmosis * p <0.05

Concentration of sulfadiazine [pg / mL] 2500 1250 500 250 125 50 25 5 IC ₅₀ [pg / m L] Proliferation 44.95 44.44 54.10 59.58 62.18 62.53 81.88 93.98 protozoa * * * ± * * ± 11.6 ± 11.6 773.97 [%] ± 9.4 2 ± 8.9 4 ± 8.2 9 11.26 ± 6.2 9 ± 11.6 5 5 5

The effect of 4- (2-fluorophenyl) -1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide (Table 6) on protozoan proliferation in cultures of L929 cells was assessed. IC50 concentration values were read from the graph made on the basis of the data included in the above table.

Table 6. The intensity of proliferation [%] of T. gondii in L929 cells cultured in the presence of 4- (2-fluorophenyl) -1- (4-methylimidazol-5-Hcarbonyl o) thiosemicarbazide in the concentration range of 1-100 pg / mL ± standard deviation

relationship 100 50 Concentration 25 [pg / mL] 10 5 1 IC50 [pg / mL] 1 26.59 * ± 3.99 73.65 ± 4.36 87.49 ± 4.32 111.23 ± 5.21 104.95 + 5.38 97.85 ± 6.02 157.59

* p <Q, Q5

On the basis of the conducted studies, it was found that 4- (2-fluorophenyl) -1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide inhibits T. gondii proliferation at a concentration expressed as IC50 lower (IC50 157.59 μg / mL) than the sulfadiazine drug used (IC50 773.97 µg / mL) (Table 7).

Table 7. Compound structure and IC50 summary

Claims (2)

1. 4- (2-fluorophenyl) -1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide, of general formula I, for use in the treatment of toxoplasmosis. 2. Use of 4- (2-fluorophenyl) -1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide, having the formula shown below, for the preparation of a medicament for the treatment of toxoplasmosis.