RU2188195C2 - 5,5'-arylidene-bis-barbituric and 5,5'-arylidene-bis-2-thiobarbituric) acid salts and 5,5'-arylidene-bis-(2-thiobarbituric) acids showing antibacterial, antiviral, immunomodulating and antitumor effect - Google Patents

Abstract

FIELD: organic chemistry, medicine. SUBSTANCE: invention relates to novel 5,5'-arylidene-bis-barbituric and 5,5'- arylidene-bis-(2-thiobarbituric) acids salts and 5,5'-arylidene-bis-(2- thiobarbituric) acids of the general formula (I)

showing an antibacterial, antiviral, immunomodulating and antitumor effect. In the general formula (I) X is taken among oxygen or sulfur atom; R¹ is taken among the group: hydrogen atom, nitro-group, alkoxy-group; R² is taken among the group: hydrogen atom, nitro-group, alkoxy-group, halogen atom; Cat^* is taken among the group including proton, pyridinium- or 2-hydroxyethylammonium cation. Compounds are preferable where: X is O; Cat⁺ is pyridinium; R¹ is H; R² is Cl (I); X is O; Cat⁺ is pyridinium; R′ is H; R² is NO₂ (II); X is O; Cat⁺ is NH₂CH₂CH₂OH; R¹ is H; R² is NO₂ (III); X is S; Cat⁺ is pyridinium; R¹ is H; R² is Cl (IV); X is S; Cat⁺ is pyridinium; R¹ is H; R² is OH (V); X is S; Cat⁺ is pyridinium; R¹ is NO₂; R² is H (VI); X is S; Cat⁺ is pyridinium; R′ = R² is MeO (VII); X is O; Cat⁺ is pyridinium;

is H; R² is Br (VIII); X is O; Cat⁺ is H; R¹ is H; R² is Cl (IX); X is O; Cat⁺ is H⁺; R¹ is H; R² is NO₂ (X). EFFECT: new compounds indicated above, valuable medicinal properties. 11 cl, 8 tbl

Description

 The invention relates to the field of organic chemistry and medicine, specifically to barbituric acids and their derivatives, and is intended for use as antiviral agents against herpes viruses and agents having antichlamydia and immunomodulatory activity.

 Many pyrimidine derivatives are biologically active substances. The spectrum of biological activity of compounds of this class is very wide. Pyrimidine derivatives are nucleic bases (uracil, thymine, cytosine), vitamins (thiamine, phosphothiamine), coenzymes (cocarboxylase), are used as pharmaceuticals with hypnotics, anticonvulsants (barbiturates, hexamidine, benzonal), diuretic (mercusal), anti-inflammatory (pentoxyl, methyluracil), antithyroid (methylthiouracil) action, are synthetic analogues of vitamins (befotiamine), anabolic (orotovy islota), anti-inflammatory and antibacterial (sulfazin, sulfadimezin, sulfamonomethoxine, sulfadimethoxine, Bactrim, salazodimetoksina), antimalarial (hloridin), anticancer (DOPA fosfemid, etimidin, fluorouracil, ftorafur, cytarabine) activity [1, 2].

 In recent decades, considerable interest has been shown in systems in which the pyrimidine ring is condensed with other heterocycles. Often, such heterocyclic systems are analogues of natural biologically active substances. These include purines, which are part of natural and synthetic biologically active substances: nucleic acids (adenine, guanine), ATP, drugs that excite the central nervous system, act on the cardiovascular system (caffeine, theobromine, theophylline, nigexin, diprofillin, xanthinol nicotinate) used to suppress tissue incompatibility during organ transplantation (azathioprine) (cytostatic and immunosuppressive effects), possessing anabolic (inosine), antileukemic (mercaptopurine) action, pyrazolo [3,4-d] irimidins used to treat diseases accompanied by hyperuricemia (allopurinol), pteridins used as diuretics (triamtren), vitamins (riboflavin, folic acid), anticancer (methotrexate) drugs, pyrimido [5,4-d] pyrimidines with vasodilating effect ( dipyridamole) [1, 2].

 The data on the biological activity of a number of derivatives of 5-ylidene barbituric acids are summarized in the review [3], in which the anticonvulsant, antimicrobial, antispasmodic, antipyretic, antitumor effects of these substances are noted. The material in this review can be supplemented with some new data on pesticidal, insecticidal [4], antipyretic [ 5,6], analgesic [7], antiviral [8-10] and fungicidal [11-13] activities shown in compounds of this group of chemicals.

 Thus, the literature data on the biological activity of 5-substituted barbituric acids and condensed systems containing a pyrimidinedione fragment have a pesticidal, insecticidal, fungicidal, anti-inflammatory, anti-tumor, antiviral, antimicrobial, anti-allergic activity, and an effect on the cardiovascular immune system. . However, the effectiveness of most already known substances is not high enough, many of them are toxic and have side effects. In addition, most microbes that cause diseases in humans and domestic animals are resistant to the main existing drugs. The indicated resistance does not allow to cope effectively with such life-threatening infections as tuberculosis, herpes, chlamydia [14-16]. The high resistance of tumor cells to existing antitumor drugs is well known [17].

 In the literature there are only a few examples of condensation of barbituric and 2-thiobarbituric acids with carbonyl compounds in a ratio of 2: 1 [18-22]. Thus, barbitucuric acid with isatin and 1-methylisatin reacts to form bisbarbiturilizatin [18], when heated with benzaldehyde in aniline, it turns into the 5.5-benzylidenebis (2-thiobarbituric) acid dianilinium salt [19] when heated 5-salicylidene barbituric acid in ethanol, it is converted, according to the authors of [19-22], into 5,5'-salicylidene bis (2-thiobarbituric) acid. However, it was shown in [23,24] that barbituric and 2-thiobarbituric acids give 5- (4,6-dioxo-5-pyrimidinyl) chromeno [2,3-d] pyrimidines with salicylic aldehydes. The synthesis of 5,5'-or-denbisbarbituric or 5,5'-ylidenebis (2-thiobarbituric) acids from aliphatic aldehydes and ketones [6,25], as well as 5,5'-benzylidenebisbarbituric acid [26] is a description of the synthesis of apparently erroneous.

 5.5'-salicylidenebis (2-thiobarbituric) acid is also known. Its chemical structure and properties are described in detail in [27]. It has low toxicity and prolongs sleep caused by barbiturates, but not by other sympathomimetics; in addition, it exhibits a vasopressor effect. No other biological activity was found in her. The last compound is the closest to the claimed in its chemical nature. Therefore, it is selected as a prototype of the invention.

 The above material demonstrates the promise of the search and creation of new effective biologically active substances by condensation of barbituric acids with carbonyl compounds, leading, in particular, to 5,5'-arylidenebis (2-thiobarbituric) acids or salts of 5,5'-arylidenebisbarbituric and 5 , 5'-arylidenebis (2-thiobarbituric) acids.

Object of the invention
The objective of the invention is the creation of a new group of chemicals with antimicrobial, antiviral, immunomodulating and antitumor activities.

где: Х выбран из группы КИСЛОРОД, СЕРА,
R¹ выбран из группы ВОДОРОД, НИТРОГРУППА. АЛКОКСИГРУППА,
R² выбран из группы ВОДОРОД, НИТРОГРУППА, ГИДРОКСИГРУППА, АЛКОКСИГРУППА, ГАЛОГЕН,
Cat⁺ выбран из группы ПРОТОН, ПИРИДИНИЙ- или 2-ГИДРОКСИЭТИЛАММОНИЙ - КАТИОН
При этом лучшие известные заявителю варианты заявляемых веществ - при:
Х=O (кислород), Cat⁺ - пиридиний, R¹=H (водород), R²=C1 (галоген) (I),
Х= O (кислород), Cat⁺ - пиридиний, R¹=H (водород), R²=NO₂(нитpoгpyппa) (II),
Х= O (кислород), Cat⁺ - Н₃N⁺СН₂СН₂ОН (2-гидроксиэтиламмоний ), R¹=H (водород), R²=NO₂ (нитрогруппа) (III);
X=S (сера), Cat⁺ - пиридиний, R¹=H (водород), R²=Cl (галоген) (IV);
X=S(cepa), Cat⁺ - пиридиний, R¹=H(водород), R²=OH (гидроксигруппа) (V),
X=S (сера), Cat⁺ - пиридиний, R¹=NO₂ (нитрогруппа), R²=H (водород) (VI),
X=S (сера), Cat⁺ - пиридиний, R¹=R²=MeO (алкоксигруппа) (VII);
Х= O (кислород), Cat⁺ - пиридиний, R¹= H (водород), R²=Br (галоген) (VIII);
Х= O (кислород), Cat⁺ - H⁺ (протон), R¹=H (водород), R²=Cl (галоген) (IX);
Х=O (кислород), Cat⁺ - H⁺ (протон), R¹=H (водород), R²=NO₂ (нитрогруппа) (X).SUMMARY OF THE INVENTION
The problem is solved by the synthesis of new substances - 5,5'-arylidenebis (2-thiobarbituric acid) and salts of 5,5'-arylidenebisbarbituric and 5 5'-arylidenebis (2-thiobarbituric) acids of the general formula

where: X is selected from the group OXYGEN, SERA,
R ^{1 is} selected from the group HYDROGEN, NITRO GROUP. Alcoxy Group,
R ^{2 is} selected from the group HYDROGEN, NITROGROUP, HYDROXYGROUP, ALCOXYGROUP, HALOGEN,
Cat ^{+ is} selected from the group PROTON, Pyridinium or 2-Hydroxyethylammonium - Cation
In this case, the best known to the applicant options for the claimed substances - with:
X = O (oxygen), Cat ⁺ - pyridinium, R ¹ = H (hydrogen), R ² = C1 (halogen) (I),
X = O (oxygen), Cat ⁺ - pyridinium, R ¹ = H (hydrogen), R ² = NO ₂ (nitro group) (II),
X = O (oxygen), Cat ⁺ - H ₃ N ⁺ CH ₂ CH ₂ OH (2-hydroxyethylammonium), R ¹ = H (hydrogen), R ² = NO ₂ (nitro group) (III);
X = S (sulfur), Cat ⁺ - pyridinium, R ¹ = H (hydrogen), R ² = Cl (halogen) (IV);
X = S (cepa), Cat ⁺ - pyridinium, R ¹ = H (hydrogen), R ² = OH (hydroxy group) (V),
X = S (sulfur), Cat ⁺ - pyridinium, R ¹ = NO ₂ (nitro group), R ² = H (hydrogen) (VI),
X = S (sulfur), Cat ⁺ - pyridinium, R ¹ = R ² = MeO (alkoxy group) (VII);
X = O (oxygen), Cat ⁺ - pyridinium, R ¹ = H (hydrogen), R ² = Br (halogen) (VIII);
X = O (oxygen), Cat ⁺ - H ⁺ (proton), R ¹ = H (hydrogen), R ² = Cl (halogen) (IX);
X = O (oxygen), Cat ⁺ - H ⁺ (proton), R ¹ = H (hydrogen), R ² = NO ₂ (nitro group) (X).

 It should be noted that the use of the remaining representatives of the alkoxy group and halogens does not lead to fundamental differences in the synthesis processes and biological properties of the claimed substances, i.e. not specific elements are essential, but their belonging to the chemical groups indicated in the general formula.

 The proposed best options for clarity are presented in table A.

 The above general formula and all of the specific compounds listed above are new; they are unknown to applicants from available sources of information.

Disclosure of invention
The invention is illustrated by the following eight examples of the synthesis of the claimed substances, three tables of physico-chemical characteristics of the obtained substances and the results of five series of experiments to determine comparative biological properties (with the corresponding tables of quantitative studies), where
Example 1 - a variant of the synthesis of the pyridinium salts of 5,5'-arylidenebisbarbituric and 5,5'-arylidenebis (2-thiobarbituric) acids (I, II, IV-VIII).

 Example 2 - a variant of the synthesis of the pyridinium salts of 5,5'-arylidenebisbarbituric and 5,5'-arylidenebis (2-thiobarbituric) acids (I, II, IV-VIII).

 Example 3 - a variant of the synthesis of 2-hydroxyethylammonium salt of 5.5 '- (n-nitrobenzylidene) bisbarbituric acid (III).

 Example 4 - a variant of the synthesis of 5,5'-arylidenebis (2-thiobarbituric) acids (IX, X).

 Example 5 is the best known to the authors version of the synthesis of the pyridinium salt of 5.5 '- (n-bromobenzylidene) bisbarbituric acid (VIII).

 Example 6 - the best known to the authors version of the synthesis of the pyridinium salt of 5.5 '- (n-chlorobenzylidene) bis (2-thiobarbituric) acid (IV).

 Example 7 - the best known to the authors version of the synthesis of 2-hydroxyethylammonium salt of 5.5 '- (n-nitrobenzylidene) bisbarbituric acid (III).

 Example 8 - the best known to the authors version of the synthesis of 5.5 '- (n-nitrobenzylidene) bis (2-thiobarbituric) acid (X).

Table 1 - PMR spectra of solutions of the obtained substances in DMSO-d ₆ (δ, ppm)
Table 2 - NMR ¹³ spectra of solutions of salts of 5,5'-arylidenebisbarbituric, 5,5'-arylidenebis (2-thiobarbituric) acids in DMSO-d ₆ (δ, ppm).

 Table 3 - Yields, decomposition temperatures, and elemental analysis of 5.5'-arylidenebis (2-thiobarbituric) acids, salts of 5.5'-arylidenebisbarbituric and 5.5'-arylidenebis (2-iobarbituric) acids.

 Experiment 1 - Determination of the effect of compounds on pathogenic mycobacteria.

 Experiment 2 - Determination of acute toxicity of the claimed compounds.

 Experiment 3 - Determination of the effect of the claimed compounds on the herpes virus.

 Experiment 4 - Determination of interferon-inducing activity of the claimed compounds.

 Experiment 5 - Determination of the effect of the claimed compounds on Chlamydia trachomatis.

 Example 1. A variant of the synthesis of the pyridinium salts of 5,5'-arylidenebisbarbituric and 5,5'-arylidenebis (2-thiobarbituric) acids (I, II, IV-VIII).

A mixture of 4.6 mmol of 5-arylidene barbituric acid and 46 mmol of barbituric acid in 10-15 ml of pyridine is refluxed for 1-2 hours. After cooling the reaction mixture, the precipitate is filtered off, washed with acetone and dried at 55-60 ^° C at a residual pressure of 30 mmHg. To isolate thio derivatives from the reaction mixture, 10-20 ml of dioxane or water are added to it, the precipitate formed is filtered off, washed with ethanol and dried.

 Example 2. A variant of the synthesis of the pyridinium salts of 5,5'-arylidenebisbarbituric and 5,5'-arylidenebis (2-thiobarbituric) acids (I, II, IV-VIII).

A mixture of 3 mmol of barbituric acid and 1.5 mmol of the corresponding aldehyde in 10-15 ml of pyridine is refluxed for 1-2 hours. After cooling the reaction mass, the precipitate is filtered off, washed with acetone and dried at 55-60 ^° C at a residual pressure of 30 mm RT .art. To isolate thio derivatives from the reaction mixture, 10-20 ml of dioxane or water are added to it, the precipitate formed is filtered off, washed with ethanol and dried. For reactions using 3 mmol of barbituric acid and 1.5 mmol of the corresponding aldehyde. Synthesis and isolation of products is carried out similarly to the previous method.

 Example 3. Variant of synthesis of 2-hydroxyethylammonium salt 5.5 '- (n-nitrobenzylidene) bisbarbituric acid (III).

2 g of the pyridinium salt of 5.5 '- (n-nitrobenzylidene) bisbarbituric acid is added to a solution of 0.4 g of 2-aminoethanol in 50 ml of ethanol. The mixture was stirred at 20 ^{° C.} for 10 minutes. The precipitated bright red precipitate is filtered off, washed with ethanol 3x40 ml, dried at 50 ^o C in vacuum. The output is quantitative.

 Example 4. Variant of synthesis of 5,5'-arylidenebis (2-thiobarbituric) acids (IX, X).

To a mixture of 1 g of 2-thiobarbituric acid and 10 ml of ethanol, a solution of 0.5 g of benzaldehyde in 10 ml of ethanol is added. After stirring the reaction mass at 20 ^o C for 20-30 minutes, the precipitate is filtered off and dried. Cleaning can be carried out by heating the precipitate in acetone, in which 5.5'-arylidenebis (2-thiobarbituric) acids do not dissolve.

Examples of the best known to the authors of the options for the synthesis of the claimed compounds of formula (I)
Example 5. The best known to the authors version of the synthesis of the pyridinium salt of 5.5 '- (n-bromobenzylidene) bisbarbituric acid (VIII).

A mixture of 4.6 mmol of 5-n-bromobenzylidene barbituric acid and 4.6 mmol of barbituric acid in 10-15 ml of pyridine is refluxed for 1-2 hours. After cooling the reaction mixture, the precipitate is filtered off, washed with acetone and dried at 55-60 ^° C with a residual a pressure of 30 mm RT. Art.

 Example 6. The best known to the authors version of the synthesis of the pyridinium salt of 5.5 '- (n-chlorobenzylidene) bis (2-thiobarbituric) acid (IV).

A mixture of 3 mmol of 2-thiobarbituric acid and 1.5 mmol of n-chlorobenzaldehyde in 10-15 ml of pyridine is refluxed for 1-2 hours. After cooling the reaction mixture, 10-20 ml of dioxane or water are added to it, the precipitate formed is filtered off, washed ethanol and dried at 55-60 ^o C.

 Example 7. The best known to the authors an example of the synthesis of 2-hydroxyethylammonium salt of 5.5 '- (n-nitrobenzylidene) -bisbarbituric acid (III).

2 g of the pyridinium salt of 5.5 '- (n-nitrobenzylidene) bisbarbituric acid is added to a solution of 0.4 g of 2-aminoethanol in 50 ml of ethanol. The mixture was stirred at 20 ^{° C.} for 10 minutes. The precipitated bright red precipitate is filtered off, washed with ethanol 3x40 ml, dried at 50 ^o C in vacuum. The output is quantitative.

 Example 8. The best known to the authors an example of the synthesis of 5.5 '- (n-nitrobenzylidene) bis (2-thio-barbituric) acid (X).

To a mixture of 1 g of 2-thiobarbituric acid and 10 ml of ethanol, a solution of 0.5 g of benzaldehyde in 10 ml of ethanol is added. After stirring the reaction mass at 20 ^o C for 20-30 minutes, the precipitate is filtered off and dried. Cleaning can be carried out by heating the precipitate in acetone, in which 5.5'-arylidenebis (2-thiobarbituric) acids do not dissolve.

Experimental verification of the biological activity of the claimed compounds
Experiment 1. Determination of the effect of compounds on pathogenic mycobacteria
The experiment used strains isolated from patients in clinics in St. Petersburg (Russia) in 1996 M. tuberculosis 61 (S), sensitive to the action of known antimicrobial agents (aminoglycosides, rifampicin, isoniazid, ethambutol), M. tuberculosis 16 ^® , stable to the main antimycobacterial drugs; the study was performed on Levenshtein-Jensen medium. Drugs dissolved in (DMSO) were added to separate tubes with medium at a final concentration of 10 mg / L. After seeding of test strains on the surface of the medium, the tubes were incubated for 30 days. DMSO at appropriate concentrations and standard preparations used to treat tuberculosis and mycobacteriosis were used in the control (table 4).

 The data obtained indicate that the claimed compounds indicated in the table inhibit the growth of all species and strains of mycobacteria used in the experiment. Thus, in vitro, they act on a strain of mycobacteria isolated from patients that is resistant to the main drugs used to treat tuberculosis. The rest of the claimed compounds have in similar conditions less antimycobacterial activity.

Experiment 2. Determination of acute toxicity of the claimed substances
The determination of acute toxicity was carried out on outbred white mice weighing 18-20 g. The inventive compounds were administered in various concentrations: 1500, 700, 500, 100, 20 and 5 mg / kg. Five animals were used to study each concentration of the compound. The drug was administered once a day through the mouth or intraperitoneally. The observation period was 14 days. On days 1, 8 and 15, animals were weighed in each group. For control, animals were used that were injected with an emulsion prepared without test compounds. For macroscopic examination of internal organs, all animals that died during the experiment and survived by the end of the experiment were autopsied. In the course of the research was not observed: weight loss, changes in behavior and appearance, as well as death of animals. According to the results of opening a macroscopic pathology from the internal organs of animals in both the experimental and control groups, it was not found. Acute toxicity (LD 50) of the claimed substance is compounds X - 1000 mg / kg

 The results obtained indicate that when taken by mouth or intraperitoneally, some of the claimed compounds at a concentration of 1000 mg / kg do not have acute toxicity to mice.

Experiment 3. Determination of the effect of the claimed compounds on the herpes virus
Antiviral activity was determined in relation to the type I herpes virus (HSV-I / Leningrad / 248/88) according to the generally accepted method (21). The viruses were grown on a transplantable Vero cell culture obtained from the cell culture bank of the Institute of Cytology of the Russian Academy of Sciences.

Test setup
To cells grown on RPMI-1640 medium with 10% calf serum and placed in wells of 96 wells, virus was added at a final concentration of 10 particles / ml and the inventive compounds dissolved in DMSO at a final concentration of 100, 10 and 1 mg / L . For each tested drug concentration, 5 independent wells were used. The plateau was incubated for 60 min at 38 ^° C in a CO2 incubator. After incubation, the virus was removed and fresh medium was added again containing the claimed compounds in the used concentrations. The results were evaluated by the presence of the cytopathogenic effect of the virus on the cells after 36 hours of incubation at 38 ^° C in a CO2 incubator.

The following controls were used in the experiment:
1. Control of cell culture (ability to normal growth).

 2. Virus control (reproductive capacity assessment).

 3. Control of antiviral activity of the antiviral drug - acyclovir.

 4. Control of compounds (toxicity of compounds).

 5. Solvent control (DMSO) for toxicity.

 To assess the cytopathic effect of the virus, the number of unchanged cells was counted in 100 fields formed by a special grid of an inverted microscope eyepiece. The results are presented in table 5.

 The results obtained indicate that all of the claimed compounds have antiherpetic activity comparable to that of the standard preparation of acyclovir.

Experiment 4. Determination of interferon-inducing activity of the claimed compounds
Induction of the synthesis of interferons by the claimed drugs was carried out on the primary culture of human lymphocytes (these cells in the human body are the main producers of interferons). To obtain a culture of lymphocytes, fresh (12 hours after collection) blood of healthy donors (not of the second group) was used. To isolate lymphocytes, heparinized blood obtained from a healthy donor was centrifuged in a density gradient of ficoll-verographin 1.71 g / cm ³ to isolate a fraction of immunocompetent cells. The specified fraction was selected and diluted with RPMI-1640 nutrient medium containing 5% fetal bovine serum, 0.3 mg / ml L-glutamine, 100 units / ml penicillin, 50 mg / ml streptomycin. The concentration of lymphocytes was taken into account after staining with methylene blue and counting the number of cells in the Goryaev chamber. Stock solutions of the claimed substances were diluted with RPMI-1640 nutrient medium so that the final concentrations of the substances were a number: 100 mg / l, 10 mg / l, 1 mg / l after application of the suspension of lymphocytes. The final concentration of lymphocytes in the induction mixture was 3x10 ⁶ cells / ml.

In parallel with the experimental samples, the following controls were affixed:
1) control of spontaneous production of interferons (IFN) by lymphocytes;
2) control of the process under the influence of a standardized IFN inducer N-methyl-N- (α-D-glucopyranosyl) ammonium-10-methylene-carboxylate acridone (cycloferon);
3) control of the process under the influence of a standardized IFN inducer - Neovir (sodium 10-methylenecarboxylate-9-acridone) with the corresponding content of DMSO in experimental samples;
4) control of the spontaneous production of interferons in the presence of DMSO in an amount corresponding to the test samples.

Control and experimental samples were incubated for 24 hours at 37 ^o C. After incubation, the samples were centrifuged at 2000 g to precipitate the cell elements and IFN-containing supernatant was taken from the samples, which were analyzed for the quantitative content of IFN. The cell pellet was resuspended in the same volume of the nutrient medium, stained with vital dye, trypan blue, and the number of cells in the Goryaev chamber was counted (as described above) to determine the cytotoxic effect of the preparations.

Quantitative determination of the content of IFN in the control and experimental samples was carried out using an enzyme-linked immunosorbent assay system on IFN-a produced by Protein circuit LLP ProCon IF2 plus. The enzyme-linked immunosorbent assay using horseradish peroxidase as an indicator enzyme was used to determine the amount of interferon in the sample. Bound peroxidase activity was measured using an automatic microplate photometer with a microprocessor at a wavelength of 450 nm. To calculate the results, IFN activity was determined in parallel for standard IFN solutions containing a known amount of the drug. Based on the results obtained, a calibration curve was constructed that, when using the microprocessor of an automatic photometer, obtain data expressed in International Activity Units (ME). The analysis results are expressed in ME IFN activity per ml in this induction system containing 3 • 10 ⁶ lymphocytes / ml. Each experimental and control point was investigated in 4 parallels.

Enzyme immunoassay controls:
1. Control of DMSO with a nutrient medium.

 2. Control of system components (according to the instructions). All results were taken into account only if the controls corresponded to the system passport data.

 The results were subjected to statistical analysis by the t-criterion and the calculation of the confidence interval at p = 0.05. An analysis of the convergence of the results in parallel experiments is performed.

 Studies have shown that virtually all of the claimed substances have the ability to induce the synthesis of IFN, which indicates their effectiveness against viral infections and antitumor activity (table 6).

Experiment 5. Determination of the effect of the claimed compounds on Chlamydia trachomatis
Antimicrobial activity of the claimed compounds was determined in relation to C. trachomatis D323 - a standard strain from the collection of the Department of Microbiology of St. Petersburg State University. ac. I.P. Pavlova. This strain was isolated from a patient with chlamydial urethritis, has a morphology and physiological activity characteristic of representatives of this species, is sensitive to the action of drugs used to treat chlamydial infection. The cell cultures MsSou and L929 obtained from the Institute of Cytology of the Russian Academy of Sciences were used in the work.

Test setup
Cells were grown in neutral glass vials in RPMI-1640 medium supplemented with 10% fetal calf serum. The experiment was set in glass (deprived of toxicity) flat-bottomed vials with coverslips. Cells were introduced into the medium at a final concentration of 1x10 cells / ml. After receiving the monolayer, standard infectious doses of chlamydia were stored in the tubes, stored in a frozen state at -70 ^o C. At the same time, the test compound was added to the cells at a final concentration of 100 mg / L. The sample was centrifuged at 2400g for 60 minutes at room temperature and incubated at 37 ^° C for 2 hours. After this, the nutrient medium was changed to a new one containing 5% fetal calf serum and cycloheximide (2 μg / ml) with repeated application of the claimed compounds in the same concentration. Samples were duplicated in parallel using a medium without cyclohexemide in order to exclude its effect on the studied substances. Samples were incubated for 48 hours in a CO2-incubator.

 The controls included: control of cell cultures, control of the action of solvents, control of the action of chlamydia in the absence of any drugs, control of the sensitivity of chlamydia to the standard antimicrobial drug - ciprofloxacin [29], control of the tested compounds for toxicity to cell cultures.

 The results were evaluated by detecting chlamydial cytoplasmic inclusions using the immunofluorescence method (MicroTrac Chlamydia trachomatis Direct Specimen Test) and chlamydial antigens using CylaMonoScreen (Russian-British Joint Venture 66 Regent's Parc Road London NW1 7SX) [30,31]. The effect of the drug was determined by analyzing the state of the monolayer and the number of cells with CPV compared with the control (cell culture infected with C. trachomatis D323), taking into account the number of unchanged cells in 100 fields of view obtained using a special microscope eyepiece grid.

 The results of control samples that meet the requirements of the experiment: control of cell culture — cell morphology and monolayer state correspond to this type of cell, control of chlamydia growth in cell culture — presence of CPV in a monolayer, control of the action of a standard antimicrobial drug — decrease in the number of CPV in a monolayer compared to the previous control , toxicity control of the claimed compounds - no toxicity, solvent control - no toxic effect on cells. The results of the tests are presented in table 7.

 The data obtained indicate that the claimed compounds are shown in table 7, have a pronounced activity against chlamydia, superior to that of the standard drug - ciprofloxacin.

 The rest of the claimed compounds have less pronounced activity to protect cells from chlamydia in the selected experimental conditions.

Industrial applicability
Examples 1-8 of the synthesis, practical identification and analysis of the claimed compounds, are shown in tables 1-3, confirm the possibility of laboratory and industrial synthesis of all ten of the claimed compounds by means developed by the modern pharmaceutical industry, as well as their clear identification by generally accepted control methods.

 The above results of 5 series of experiments on the determination of biological activity showed that the claimed compounds have biological activity against various microorganisms, including mycobacteria, chlamydia, herpes simplex virus, as well as interferon-inducing activity. The latter indicates the possibility of their use in the treatment of herpes, other viral and some cancers.

 The above facts prove the achievement of the objectives set by the invention: a new class of heterocyclic compounds with high and wide biological activity, in particular, antimycobacterial, immunostimulating, antichlamydial and antiviral, has been synthesized.

 Thus, in our opinion, the claimed means (substances) satisfy all the requirements for the invention: they are new, non-obvious and industrially applicable.

Literature
1. Pharmaceutical microbiology, Ed. by WB Hugo and AD Russel Blackwell Scientific Publications, Oxford, 1987, 511 p.

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Claims (11)

1. Salts of 5,5'-arylidenebisbarbituric and 5,5'-arylidenebis (2-thiobarbituric) acids and 5,5'-arylidenebis (2-thiobarbituric) acids of the general formula

where X is selected from oxygen or sulfur;
R ^{1 is} selected from the group: hydrogen, nitro group, alkoxy group;
R ^{2 is} selected from the group: hydrogen, nitro group, alkoxy group, halogen;
Cat ^{+ is} selected from the group: proton, pyridinium or 2-hydroxyethylammonium cation,
possessing antimicrobial, antiviral, immunomodulating and antitumor effects. 2. A substance according to claim 1, characterized in that X = O, Cat ⁺ is pyridinium, R ¹ = H, R ² = Cl (I). 3. The substance according to claim 1, characterized in that X = O, Cat ⁺ - pyridinium, R '= H, R ² = NO ₂ (II). 4. The substance according to claim 1, characterized in that X = O, Cat ⁺ - NH ₂ CH ₂ CH ₂ OH, R ¹ = H, R ² = NO ₂ (III). 5. The substance according to claim 1, characterized in that X = S, Cat ⁺ is pyridinium, R ¹ = H, R ² = Cl (IV). 6. The substance according to claim 1, characterized in that X = S, Cat ⁺ is pyridinium, R ¹ = H, R ² = OH (V). 7. The substance according to claim 1, characterized in that X = S, Cat ⁺ is pyridinium, R ¹ = NO ₂ , R ² = H (VI). 8. The substance according to claim 1, characterized in that X = S, Cat ⁺ is pyridinium, R ¹ = R ² = MeO (VII). 9. The substance according to claim 1, characterized in that X = O, Cat ⁺ is pyridinium, R ¹ = H, R ² = Br (VIII). 10. The substance according to claim 1, characterized in that X = O, Cat ⁺ —H, R ¹ = H, R ² = Cl (IX). 11. The substance according to claim 1, characterized in that X = O, Cat ⁺ -H ⁺ , R ¹ = H, R ² = NO ₂ (X).

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