PL237491B1 - 4-bromophenyl-1-(4-methylimidazol-5-yl)thiosemicarbazide derivatives and method of their preparation and medical use - Google Patents

Abstract

The subject of the application are derivatives of 4-bromophenyl-1-(4-methylimidazol-5-yl)thiosemicarbazide, with the general formula shown in the drawing, where the bromo substituent is assigned to the ortho, meta, or para position, respectively. The application also covers the use of the aforementioned compounds and a method for their preparation. This method involves reacting 4-methyl-5-imidazole-5-carboxylic acid hydrazide with isothiocyanate in a 1:1 molar ratio. The reaction is carried out in anhydrous ethanol at the solvent's boiling point. The resulting product is cooled, the precipitate is filtered off, and after drying, it is crystallized.

Description

The present invention relates to 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide derivatives where the bromo substituent is in the ortho, meta or para position, respectively, their preparation and medical use.

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. In turn, Walchshofer et al. [Bioorg Med Chem 2000, 10, 871] described a series of condensed furanoisoquinoline derivatives which, at a concentration of 2 μg / 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.

Toxoplasma gondii is a cosmopolitan parasite that causes toxoplasmosis in all species of mammals, 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. Combination therapy consists of 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 only exception are infected pregnant women under the 6th month of pregnancy who are administered spiromycin showing high affinity to the placenta, but unfortunately weak 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 hematological symptoms (megaloblastic anemia, thrombocytopenia, leukopenia) and neurological disorders (headache and dizziness, ataxia, seizures, insomnia and depression), or changes in the skin and mucous membranes (dermatitis, abnormal pigmentation, rash, glossitis). The following 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 Res 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].

The applicants have carried out research aimed at obtaining thiosemicarbazide derivatives not described in the literature, whose antiparasitic activity against the pathogenic parasite Toxoplasma gondii would exceed that of the known compounds of this group. The obtained results show a strong antiparasitic activity against T. gondii of the derivatives according to the invention, which is important in the range of non-toxic concentrations. The antiparasitic activity of the compounds according to the invention is many times higher than that of the present day

The sulfadiazine drug was used. The obtained data allow to expect that the tested compounds can be the starting compounds in the search for new drugs to fight toxoplasma.

As can be seen from the above facts, the use of classic drugs for the treatment of toxoplasmosis is associated with considerable risk. 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% of the respondents went above this norm. There is therefore a real need to develop new drugs capable of effectively treating toxoplasmosis without the side effects discussed above.

The compounds of the invention are derivatives of 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide (1,2,3 in the following tables) with the general formula shown in the figure, where the bromo substituent is assigned to the ortho, meta or couple.

The method of obtaining the derivatives according to the invention consists in reacting 4-methyl-5-imidazole-5-carboxylic acid hydrazide with isothiocyanate in a molar ratio of 1: 1. The reaction is carried out in anhydrous ethanol at the boiling point of the solvent, preferably when the reaction time is from 10 to 30 min. The progress of the reaction is monitored by thin layer chromatography. After the product has formed, the contents of the flask are cooled, the resulting precipitate is filtered off and crystallized from 96% ethanol after drying.

The invention also relates to 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide derivatives having the general formula shown in the figure, where the bromo substituent is assigned ortho, meta or para positions, respectively, for use in the treatment of toxoplasmosis.

The use of 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide derivatives with the general formula shown in Figure 1, where the bromine substituent is assigned to the ortho, meta or para position, respectively, for the preparation of a preparation intended for the treatment of toxoplasmosis.

The compounds of the invention show strong inhibition of T. gondii proliferation in vitro and no significant cytotoxicity to the host cells. Thus, the compounds obtained according to the invention may find use for the preparation of new drugs in the treatment of toxoplasmosis.

Assessment of the effect of 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide derivatives 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 for in 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

L929 cells - ATTC-Catalog No. CCL-1 ™ at a density of 1x104 / 100 μL / 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 added to it. 100 μL of the respective dilutions of the test compound 1,2 and 3 (0.0; 3.91; 7.81; 15.63; 31.25; 62.50; 125.00) and, for comparison, sulfadiazine (Sigma), which final concentrations 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 µL of MTT (C = 1 mg / mL, Sigma) was added to each well of the plate and incubated (37 ° C and 5% CO 2). After 2 h the plates were centrifuged (200 x g, 10 min) and the formazan crystals were dissolved by adding 150 µL DMSO / well. The color of the product was stabilized by adding 25 μL of glycine buffer each. The absorbance level was measured with an ELISA reader at a wavelength of λ = 550 nm.

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The significance level p was determined by the non-parametric Mann-Whitney U test using the SigmaStat program. The results at the 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 test compounds on the viability of the L929 host cells by means of the MTT assay. Cell cultures incubated in IMDM medium without addition of test compounds and solvent (DMSO) were used as controls. Cell viability was calculated by the formula:

Wmoartsorbancf of the test sample _{χ 1 <J0X}

Absorbed 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% cell viability of the L929 lineage were selected to study the effect of compounds on intracellular proliferation of protozoa.

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

Due to the necessity to dissolve the compounds 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 did not significantly affect the viability and morphology of L929 cells (Tab. 1).

Ta b. 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 (Tab. 2). During microscopic analysis, no changes in the morphology of cells incubated with various concentrations of a commercially available drug were observed.

Ta b. 2

Cell viability [%] of the L929 line at individual sulfadiazine concentrations ± standard deviation

Concentration of sulfadiazine [pg / mL] 2500.0 1250.0 500.0 125.0 50.0 25.0 5.0 CC30 [pg / mL] living cells 81.01 83.28 94.5 95.3 94.5 91.9 81.2 > 2500 [%] ± 5.46 ± 2.56 9 4 9 9 9 ± 3.5 ± 4.7 ± 4.5 ± 3.2 ± 4.1 9 9 6 1 3

The effect of compounds on the viability of L929 cells was investigated. CC30 concentrations were determined for the L929 cell line (Tab. 3) and photos of the cell culture were taken with the addition of the determined concentration of the tested compound (1,2, 3).

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Ta b. 3 Cell viability [%] of the L929 lineage in the concentration range of 1,4-disubstituted thiosemicarbazide derivatives 1-125 pg / mL ± standard deviation

relationship Compound concentration [pg / mL] cc ₃₀ [pg / mL] 125.00 62.50 31.25 15.63 7.81 3.91 1 86.39 ± 5.89 78.58 ± 3.56 80.47 ± 3.39 87.26 ± 3.59 84.58 ± 4.64 97.23 ± 5.55 2 71.93 ± 2.56 71.59 ± 5.69 71.74 ± 5.21 83.72 ± 3.55 86.65 ± 2.99 95.06 ± 3.03 > 125 3 86.06 ± 3.59 86.89 ± 3.58 82.30 ± 2.47 92.42 ± 5.48 92.27 ± 2.69 95.50 ± 3.69

On the basis of the conducted research, it was found that the derivatives of 4-bromophenyl-1- (4-methylimidazol-5-yl carbonyl) thiosemicarbazide (1, 2, 3) do not show cytotoxicity in the concentration range of 0.0-125 μg / mL. The tested thiosemicarbazide derivatives above the concentration of 125 pg / mL precipitate in the form of crystals during the course of the experiment, which makes it impossible to accurately determine the CC30 value.

Study of the influence of 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide derivatives on the proliferation of T. gondii in L929 cells (irritated uracil incorporation test).

The effect of 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide (1, 2, 3) derivatives on the intracellular proliferation of T. gondii BK strain in L929 cells was investigated with the tritium-labeled uracil incorporation test. 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 IC50 value (inhibitory concentration 50 - the concentration causing 50% inhibition of protozoan multiplication) 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 (1x10 ^4/50 mL / well) were infected with T. gondii strain tachyzoitami AB in the ratio 1 cell host 10 protozoan cells (1x10 ^5/50 mL / well). Microcultures were incubated for 2 h 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 (1,2 or 3) to give final concentrations: 0.0; 3.91; 7.81; 15.63; 31.25; 62.50; 125.0 μg / mL, 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 μΟί / 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 at the 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 influence of the solvent on the proliferation of T. gondii in L929 cells was examined (Tab. 4).

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Ta b. 4

T. gondii proliferation intensity 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

* /) <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 tested compound was microculture with sulfadiazine, a drug that has been used for a long time in the treatment of toxoplasmosis (Tab. 5). Table 5 shows the data on the activity against T. gondii to compare the activity of the compounds 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 compounds according to the invention show higher efficacy than the known drug.

Ta b. 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 44.9 44.4 54.1 59.5 62.1 62.53 81.8 93.9 protozoa 5 * 4 * about* 8 ± 8 * * 8 8 773.9 [%] ± 9.4 ± 8.9 ± 8.2 11.2 ± 6.2 ± 11.6 ± 11, ± 11, 7 2 4 9 6 9 5 65 65

The effect of 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide derivatives (Tab. 6) on the proliferation of protozoa in cultures of L929 cells was assessed. IC50 concentration values were read from the graphs made on the basis of the data included in the above-mentioned table.

Ta b. 6

Intensity of proliferation [%] of T. gondii in L929 cells cultured in the presence of 4-bromophenyl-1- (4-methylimidazol-5-yl carbonyl] thiosemicarbazide derivatives in the concentration range of 1-100 pg / mL ± standard deviation

relationship 100 50 Concentration 25 [pg / mL] 10 5 1 IC ₅₀ [pg / mL] 1 25.82 * 30.26 * 31.02 * 38.96 * 80.44 95.53 11 Gi 1 ± 4.98 ± 3.72 ± 4.76 ± 5.23 ± 6.28 ± 5.01 JL 13.84 * 23.25 * 32.90 * 35.05 * 67.08 * 107.84 11 cn WITH ± 4.62 ± 6.27 ± 5.11 ± 7.23 ± 6.33 ± 6.43 25.97 * 27.00 * 27.61 * 33.23 * 75.74 105.49 14 77 ± 5.73 ± 5.33 ± 6.34 ± 6.99 ± 4.99 ± 6.50 14, / Z

* p <0.05

On the basis of the conducted studies, it was found that the derivatives of 4-bromophenyl-1- (4-methylimidazol-5-yl carbonyljthiosemicarbazide (1, 2 and 3) show inhibition of T. gondii proliferation at lower concentration expressed as IC50 (respectively: 13.85; 11). , 50 and 14.72 pg / mL) than the sulfadiazine drug used (IC50 773.97 pg / mL) (Tab. 7).

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Ta b. 7

Structure of compounds and ICso - summary

Relationship structure Relationship name IC 50 [pg / mL] Q? ΛΛ X of ζ-ω— (\ —z x II \ ___ / o sulfadiazine 773.97 O Η H Br 1 13.85 55.9χ lower dose O Η H. 11.50 έΑχχτ Ή 2 67.3 x lower dose About Η Η 14.72 \ ^ΒΓ 3 52.6x lower dose

Example:

A mixture of hydrazide (0.01 mol; 1.40 g) and 2-bromophenylisothiocyanate (0.01 mol; 2.14 g) was heated in a round bottom flask equipped with a reflux condenser at the reflux temperature of the solvent. The reaction time was 10 min. After the product was formed, the contents of the flask were cooled, the resulting precipitate was filtered off, and after drying it was recrystallized from 96% ethanol. There were obtained 3.33 g (94.07% of theoretical yield, Tab. 8) 4- (2-bromophenyl) -1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide with a melting point of 205-207 ° C (Tab. 9 ). Proper construction of the compound and its purity was confirmed by analysis of the ¹ H NMR spectrum (Tab. 9).

Ta b. 8

Time and yield of the reaction for the preparation of 4-bromophenyl-1- (4-methylimidazol-5-yl carbonyl] thiosemicarbazide derivatives

relationship no relationship name reaction time [min] efficiency [g =%] 1 4- (2-bromophenyl) -1 - (4- methyl loimidazole -5- amount of carbonylojthiosemicarbazide 10 3.33 g = 94.07% 2 4- (3-bromophenyl) -1 - (4- methyl loimidazolo -5 - amount of carbonyl) thiosemicarbazide thirty 2.90 g = 81.92% 3 4- (4-bromophenyl) -1 - (4- methyl loimidazole-5 - amount of carbonyl) thiosemicarbazide twenty 2.75 g = 77.68%

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Ta b. 9

Physicochemical parameters of 4-bromophenyl-1- (4-methylimidazol-5-yl carbonyl) thiosemicarbazide derivatives

relationship name physicochemical parameters Ή NMR (DMSO- ^) δ (ppm): top temp. [° C] 4- (2-bromophenyl) -1- (4-methylimidazol-5-yl carbonyl) thiosemicarbazide 2.456 (s, 3H); 7,117-7,147 (m, 1H); 7.340-7.396 (m, 1H); 7.626 (s. 2H); 7.752-7.778 (d, 1H); 9.282 (s. 1H); 9,749 (s. 1H); 9.903 (s. 1H); 12,417 (s, 1H) 205-207 4- (3-bromophenyl) -1- (4-methylimidazol-5-yl carbonyl) thiosemicarbazide 2.451 (s, 3H); 7.233-7.316 (m, 1H); 7.525-7.545 (m, 1H); 7.624 (s. 2H); 7,820 (s. 1H); 9,697 (s. 1H); 9.787 (s. 1H); 9.852 (s, 1H) 12.4O3 (s, 1H) 204-206 4- (4-bromophenyl) -1- (4-methylimidazol-5-yl carbonyl) thiosemicarbazide 2.448 (s. 3H); 7.483 (s, 4H) 7.619 (s. 1H); 9,644 (s, 1H); 9,775 (s, 1H); 9.852 (s. 1H); 12,395 (s, 1H) 228-230

Patent claims

Claims (6)

CLAIMS 1. 4-Bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide derivatives having the general formula shown in the figure, where the bromo substituent is assigned ortho, meta or para positions, respectively. 2. A method for the preparation of new 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide derivatives, characterized in that 4-methyl-5-imidazole-5-carboxylic acid hydrazide is reacted with isothiocyanate, whereby the reaction is carried out in a molar ratio of 1: 1, the reaction is carried out in anhydrous ethanol at the boiling point of the solvent, the product obtained is cooled, the precipitate is filtered off, and after drying it crystallizes. 3. The method according to p. The process of claim 2, wherein the reaction time is 10 to 30 min. 4. The method according to p. A process according to claim 2, characterized in that the product obtained is crystallized from 96% ethanol. 5. 4-Bromophenyl-1- (4-methylimidazol-5-yl carbonyl) thiosemicarbazide derivatives having the general formula shown in the figure, where the bromo substituent is assigned ortho, meta or para positions, respectively, for use in the treatment of toxoplasmosis. 6. The use of 4-bromophenyl-1- (4-methylimidazol-5-ylcarbonyl) thiosemicarbazide derivatives with the general formula shown in Figure 1, where the bromine substituent is assigned to the ortho, meta or para position, respectively, for the preparation of a preparation intended for the treatment of toxoplasmosis.