PL235166B1 - 4-(3-fluorophenyl)-1-(4-methylimidazol-5-yl)thiosemicarbazide, method of preparation and medical use - Google Patents

Abstract

The subject of the application is 4-(3-fluorophenyl)-1-(4-methylimidazol-5-yl)thiosemicarbazide of the general formula shown in the drawing. The application also covers the use of the above-mentioned compound and the method of its preparation, which consists in reacting 4-methyl-5-imidazole-5-carboxylic acid hydrazide with isothiocyanate, wherein 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, preferably when the reaction time is 30 minutes, then the obtained product is cooled, the precipitate is filtered off, and after drying it is crystallized.

Description

Description of the invention

The present invention relates to a 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide of the general formula shown in the drawing, a method of preparation and a medical use 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 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 (Bioorg Med Chem Lett 2004, 14, 2773). A series of thiazolidinone and thiosemicarbazone derivatives has been described by Goes et al. (Bioogr Med Chem Lett 2005, 15, 2575). Unfortunately, among the tested compounds, only selected derivatives showed biological activity higher than that of sulfadiazine or hydroxycarbamide.

The patent description PL 212639 describes a method of obtaining a 1,4-disubstituted thiosemicarbazide derivative and a method of its preparation by reacting indole-2-carboxylic acid hydrazide with 4-nitrophenylisothiocyanate. In turn, from the description PL 214847 there are known 1- (1-phenyimidazolidine-2-ylidene) 4-substituted thiosemicarbazide derivatives and a method of their preparation by reacting benzyl isothiocyanate with appropriately substituted free 1-aryl-2-hydrazonoimidazolidine derivatives obtained from appropriately substituted hydroiodides in the presence of an organic solvent.

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 infected with this parasite. In Poland, infection occurs in 50-60% of people (Int J Parasitol 2000, 30, 1217; Mikrobiol Med 1996, 1, 14; AnnAgric 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 infection 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 6 months of pregnancy who are administered spiromycin, which has high affinity for the placenta, but unfortunately has a 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). 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 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).

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As can be seen 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 gg / 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 respondents exceeded this norm. There is therefore a real need to develop new drugs capable of effectively treating toxoplasmosis and devoid of the side effects discussed above.

The object of the invention is to obtain thiosemicarbazide derivatives whose antiparasitic activity against the pathogenic parasite Toxoplasma gondii exceeds that of the known compounds of this group.

The invention is based on 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide having the general formula shown in the figure.

A compound of the invention having the formula shown in the drawing for use in the treatment of toxoplasmosis.

The use of a compound according to the invention for the preparation of a preparation intended 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, the compound obtained according to the invention may find use in the preparation of new drugs in the treatment of toxoplasmosis.

The obtained results indicate a strong antiparasitic activity against T. gondii, which is important in the range of non-toxic concentrations. The antiparasitic activity of the compound according to the invention is many times higher than the activity of the currently used drug sulfadiazine. The obtained data allow us to expect that the tested compound may be the starting compound in the search for new drugs to fight toxoplasma.

Assessment of the effect of 4- (3-fluorophenyl) -1- (4-methylimidazol-5-iIo) carbonylthiosemicarbazide on the viability of L929 cells - MTT test

Principle of the MTT test

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

In a 96-well plate (Nunc ^TC ), L929 cells - ATTC-Catalog No. CCL-1 ™ at a density of 1 × 104/100 gL / well, suspended in IMDM (Iscove's Modified Dulbecco's Medium - CytoGen) culture medium and incubated (37 ° C, 10% CO2) for 24 h. After incubation, the medium was removed from the cells and 100 gL of the respective 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 gg / 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 gL 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 hours, the plates were centrifuged (200 x g, 10 min) and the formazan crystals were dissolved by adding 150 gL DMSO / well. The color of the product was stabilized by adding 25 gL of glycine buffer each. The absorbance level was measured with an ELISA reader at a wavelength of λ = 550 nm.

The significance level p was determined with 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. Values

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CC30 (cytotoxic concentration 30 - the concentration causing a cytotoxic effect for 30% of the tested cells) was 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 a 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 L929 cells were selected to study the effect of a compound on intracellular proliferation of the 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 cell survival curve [%] on the compound concentrations used and sulfadiazine [pg / mL], calculated on the basis of the data presented in Tab. 1 and Tab. 2, respectively.

Due to the necessity to dissolve the compound in DMSO, in the first stage of the experiment, the influence of the applied solvent concentrations on the viability of L929 cells was investigated. It was found that the concentrations of DMSO used in the experiment did not have a significant effect on the viability and morphology of L929 cells (Tab. 1).

Tab. 1 Cell viability [%] of the Union L929 in 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

* p <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 pg / mL (Tab. 2). During microscopic analysis, no changes in the morphology of cells incubated with various concentrations of a commercially available drug were observed.

Tab. 2. Cell survival [%] 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] 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 (Tab. 3) and photos of the cell culture with the addition of the determined concentration of the tested compound were taken.

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Tab. 3 Cell survival [%] of the L929 line in the concentration range of 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) car-

bonylotiosemicarbazide 1-125 μς / ιηΙ_ ± standard deviation relationship 125.00 Compound concentration [pg / mL] 62.50 31.25 15.63 7.81 3.91 CC30 [gg / mL] 1 73.62 ± 2.59 74.08 76.66 86.49 ± 3.06 ± 3.54 ± 4.21 88.86 ± 4.18 91.07 ± 3.64 > 125

On the basis of the conducted research, it was found that 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide does not exhibit cytotoxicity in the concentration range of 0.0-125 μο / πΊΐ. in the form of crystals during the experiment, which makes it impossible to accurately determine the CC30 value.

Study of the influence of 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide on the proliferation of T. gondii in L929 cells (irritated uracil incorporation test).

The effect of 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide on the intracellular proliferation of T. gondii BK protozoan in L929 cells was investigated 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 (the measured value of proliferation was taken as 100%). The IC50 value (inhibitory concentration 50 - concentration which inhibits protozoan multiplication by 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 that the protozoan multiplication is inhibited in the microculture.

Experimental part

24-hour culture of L929 cells (1 χ 10 ^4/50 μι? Well) were infected with T. gondii strain tachyzoitami AB in the ratio 1 cell host 10 cells of protozoa (1χ10 ^5/50 μι? Well). The microcultures were incubated for 2 h at 37 ° C, 10% CC> 2, allowing the protozoa to enter cells and form a parasitic vacuole. After this time, 100 µL each of: i) test compound was added to give final concentrations: 0.0; 3.91; 7.81; 15.63; 31.25; 62.50; 125.0 pg / 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. The plates were incubated for 48 h (37 ° C, 10% CC> 2), then ³ H-uracil (1 μθί / 50 μΙ) was added to each well and incubation was continued for 24 h under the conditions as above. After this time, the plates were frozen at -20 ° C. Before 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 with 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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Tab. 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

* /> <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 been used for a long time in the treatment of toxoplasmosis (Tab. 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.

Tab. 5 Intensity of proliferation [%] of T. gondii in L929 cells cultured in the presence of sulfadiazine, a commercial drug used in the treatment of toxoplasmosis * /> <0.05

Concentration of sulfadiazine [pg / mL] 2500 1250 500 250 125 50 25 5 IC ₅₀ [pg / m L] Protozoan proliferation [%] 44.95 * ± 9.42 44.44 * ± 8.94 54.10 * ± 8.29 59.58 ± 11.26 62.18 * ± 6.29 62.53 * ± 11.6 5 81.88 ± 11.6 5 93.98 ± 11.6 5 773.97

The influence of 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide (Tab. 6) on the proliferation of protozoa in cultures of L929 cells was assessed. IC50 concentration values were read from the graph made on the basis of the data contained in the above table.

Tab. 6 Intensity of proliferation [%] of T. gondii in L929 cells cultured in the presence of 4- (3-fluorophenyl) 1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide in the concentration range 1–100 pg / mL ± standard deviation

relationship 100 50 Concentration 25 [pg / mL] 10 5 1 IC50 [pg / mL] 1 43.92 * ± 6.43 54.45 * ± 4.67 63.43 * ± 4.21 96.33 ± 8.23 86.40 ± 8.39 91.55 ± 3.67 101.88

* /> <0.05

On the basis of the conducted research, it was found that 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide inhibits proliferation of T. gondii at a concentration expressed as IC50 lower (IC501.88 pg / mL) than the drug sulfadiazine ( IC50 773.97 pg / mL) (Table 7).

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The invention also relates to a process for the preparation of 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide, which consists in reacting 4-methyl-5-imidazole-5-carboxylic acid hydrazide with isothiocyanate in 1: 1 molar ratio. The reaction is carried out in anhydrous ethanol at the boiling point of the solvent, preferably the reaction time is 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 precipitate formed is filtered off and, after drying, crystallized preferably from 96% ethanol.

Example

A mixture of hydrazide (0.01 mol; 1.40 g) and 3-fluorophenylisothiocyanate (0.01 mol; 1.37 g) was heated in a round bottom flask equipped with a reflux condenser at the reflux temperature of the solvent. The reaction time was 30 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 2.36 g (80.55% of theory, Tab. 8) 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide with a melting point of 191-192 ° C (Tab. 9) . Proper construction of the compound and its purity was confirmed by analysis of the ¹ H NMR spectrum (Tab. 9).

Tab. 8 Time and efficiency of the reaction for the preparation of 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) -carbonylthiosemicarbazide

no relationship relationship name response time [min] performance [g -%] 1 4- (3-fluorophenyl) -1- (4-methylimidazole- 5-yl) carbonylthiosemicarbazide thirty 2.36 g = 80.55%

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Tab. 9 Physicochemical parameters of 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide

relationship name physicochemical parameters H NMR (DMSO-rf _e ) δ (ppm): top temp. [° C] 4- (3-fluorophenyl) -1- (4-methylimidazole- 5-yl) carbonylthiosemicarbazide 2.425 (s. 3H); 6,911-6,966 (m, 1H); 7.307-7.342 (m, 2H); 7.528 (s. 1H); 7.627 (s. 1H); 9,698 (s. 1H); 9.813 (s, 1H); 9.855 (s. 1H); 12,400 (s, 1H) 191-192

Claims (5)

Patent claims 1. 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide, having the general formula shown in the figure. 2. The method for the preparation of 4- (3-fluorophenol) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide of the general formula shown in the figure, characterized in that 4-methyl-5-imidazole-5-carboxylic acid hydrazide is subjected to reaction with isothiocyanate, the reaction being carried out in a molar ratio of 1: 1 in anhydrous ethanol at the boiling point of the solvent, preferably when the reaction time is 30 minutes, then the product obtained is cooled, the precipitate is filtered off and crystallized after drying. 3. The method according to p. A process according to claim 2, characterized in that the product obtained is crystallized from 96% ethanol. 4. 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide of the formula for use in the treatment of toxoplasmosis. 5. Use of 4- (3-fluorophenyl) -1- (4-methylimidazol-5-yl) carbonylthiosemicarbazide of the formula shown in the figure for the preparation of a preparation intended for the treatment of toxoplasmosis.