2,4-dioxo-5-arylidene-1,3-pyrimidine

 

(57) Abstract:

The invention relates to novel 2,4-dioxo-5-arylidene-1,3-pyrimidines of General formula I, where R is independently selected from the group: H, HE, lowest alkoxyl, halogen, nitro, di(lower)alkylamino, n = 1-3, or two adjacent R along the benzene ring to which they are attached, when n = 2, 4 form a benzo, and dibenzo when n = 2 form a 3,4-dioxolane ring. These compounds have antiviral, immunostimulant, antichlamydial, TB, psychogenesis, analgesic and hepatoprotective effect and can find application in the pharmaceutical industry. Most preferred are compounds of General formula I, where R = H, n = 1, or R = 4-CL, n = 1, or R = 3-CL, n = 1, or R = 2-CL, n = 1, or R = 4-Br, n = 1, or R = 4 HE, n = 1, or R = 2,4-(OH)2n = 2 or R = 2-OH, 5-Br, n = 2 or R = 2 Oh,5-CL, n = 2 or R = 4-F, n = 1, or R = 2,4-Cl2n = 2 or R = 2-OH, 3,5-Cl2n = 3 or R = 2-HE, 3,5-CH2n = 3 or R = 4-och3n = 1, or R = 4-one, 3-och3n = 2 or R = 4-NO2n = 1, or R = 3-NO2n = 1 or R = 2-NO2n = 1 or R = 2-OH, 5-NO2n = 2 or R = 2-HE, 5,6-benzo, n = 3, or R = 3,4-dioxolane, n = 2 or R = 2-NO2, 4,5-dioxolane, n = 3, or R = 2,3,5,6-dibenzo, n = 4 or R = 2-OH, 3,5-I2n = 3 or R = 4-N(armacology, specifically to synthetic biologically active derivative of pyrimidine.

The inventive substances are pronounced antiviral, immunostimulant (interferon-inducing), antichlamydial, TB, psychogenesis, analgesic and hepatoprotective effect.

The connectors are designed primarily for use in medical practice for the treatment of viral infections, infections caused by chlamydia, diseases associated with immunodeficiency, in particular malignant tumors, as well as tuberculosis. In addition, these compounds may be used for the same purposes in veterinary medicine.

As you know, one of the most serious problems of modern medicine are microbial and viral diseases, many of which are extremely difficult to treat, due to the lack of effectiveness of existing drugs and rapid variability of microbes, leading to the emergence of resistant forms [1, 2, 8].

Often viral diseases occur on the background of reducing the activity of the immune system of the body and are accompanied by secondary infections, this also applies to cancer. The poet is the means of treating immunodeficiencies different origin.

Known antiviral drugs can be divided into 2 groups according to the types of mechanisms of their action. The effects of the first group is associated with suppression of the reproduction of viruses in the body [1]. Antiviral drugs of the second group have an effect not so much due to exposure to the virus, much due to the stimulation of immune protection and enhance the production of endogenous interferon [2]. Interferons and their inducers are also used for treatment of several neoplastic diseases [3]. The inventive substances are products in the second group.

Chemical analogues of the claimed compounds can be R-aminouracil the following General formula

< / BR>
where R = phenyl, 2-were, 3-were, 4-were, 4-bromophenyl, 2,4-dimetilfenil, 2,5-dimetilfenil, 2,6-dimetilfenil, 2-chlorophenyl, 3-chlorophenyl, 1-naphthyl, 2-naphthyl, cyclohexyl. They are described, for example, in [9].

The closest in chemical nature to the claimed is- 6-[[3-[4-(2-Methoxyphenyl)-1-piperazinil] propyl] -amino] -1,3-dimethyluracil. Its pharmaceutical name - urapidil.

< / BR>
6-[[3-[4-(2-Methoxyphenyl)-1-piperazinil]propyl]-amino]-1,3-dimethyluracil - has a hypotensive effect. In bases [10].

Unfortunately, the spectrum of its biological activity is relatively narrow.

The task of the invention to provide new chemical compounds with broader biological activity, including antiviral activity against herpes simplex viruses), immunostimulatory activity (due to the induction of the production of endogenous interferon in the body), antimicrobial activity, psychogenesis, analgesic and hepatoprotective actions. In other words, the object of the invention is limited to chemical synthesis of biologically active substances exceeding the prototype biological activity, as well as the breadth of the action.

The problem is solved by the synthesis of new substances - 2,4-dioxo-5-arylidene-1,3-pyrimidines of General formula

< / BR>
where R is selected from the group: H, HE, alkoxyl, halogen, nitro, dialkyl, aryl and 3,4-dioxolane.

List of all 25 synthesized and identified substances are given in table. 1.

Listed in the table. 1 new substances, because they are not known from the available sources of information.

The availability of a wide range of effective biological activity of the claimed substances not ViceChairmen explain the following:

- how to retrieve all 25 compounds I-XXV;

example synthesis of 2,4-dioxo-5-(2-hydroxy-3,5-dichlorobenzamide)imino-1,3-pyrimidine;

data PMR spectroscopy of compounds I-XXV (PL. 2);

- the experimental data for the determination of the biological activity of the inventive compounds in comparison with the known effective date means the same purposes, namely:

experiment 1 - determination of the validity of the claimed compounds on herpes simplex virus (PL. 3);

experiment 2 - determination of the interferon-inducing activity of the claimed compounds (with table. 4);

experiment 3 - determining steps of the inventive compounds on Cnlamydia_ trachomatis (table. 5);

experiment 4 - determination of antimicrobial activity of compounds (with table. 6);

experiment 5 - definition psychoparasite action of the claimed compounds (with table. 7);

experiment 6 - evaluation of analgesic activity of the claimed compounds (with table. 8);

experiment 7 - hepatoprotective action of the claimed compounds (with table. 9);

experiment 8 - determine the maximum tolerated dose (table. 10).

The method of obtaining the compound (I-XXV.

Target 2,4-dioxo-5-arylidene-1,3-pyrimidines receive the comfort of a mixture of ethanol - water 1:1. When boiling a mixture of the aldehyde with aminouracil falls colorless crystalline residue.

The products are obtained from the outputs of 45-95% of theoretical.

Example of synthesis of 2,4-dioxo-5-(2-hydroxy-3,5-dichlorobenzamide)imino-1,3-pyrimidine.

The flask 1.27 g of 5-aminouracil, 150 ml of water. The mixture is heated under stirring until complete dissolution of the precipitate. In parallel, in 50 ml of ethanol is dissolved 1.91 g of 3,5-dichlorosalicylic aldehyde and added to a solution of 5-aminouracil. Immediately precipitation bright orange color. The reaction mixture boils under stirring for 1 h and 1 h stirring is continued at room temperature. Then the reaction mixture is left overnight. The precipitate is filtered off, washed with warm water, alcohol, and dried. The product yield is 92%.

The rest of the claimed compounds are synthesized similarly using the other R

A noticeable difference in the yield of the target product.

Compounds of General formula are colorless or light-colored crystalline substance, soluble in dimethyl sulfoxide, pyridine. The melting temperature of all substances exceed 300oC.

Well the th carbon - isopropanol = 9:1. The structure of synthesized compounds proved by the method of PMR spectroscopy.

Data PMR spectroscopy of compounds I-XXV shown in table. 2.

In the PMR spectra degenerate characteristic signal of the two protons of aminogroup in brazilina ring (PL. 2 presents the average value of chemical shift).

The experimental data for the determination of the biological activity of the claimed compounds.

Experiment 1. The definition of the action of the claimed compounds on herpes simplex virus.

Antiviral activity was studied against herpes simplex virus type I (HSV-I /Leningrad/248/88) by the conventional method [5]. Viruses were grown on transplantable cell culture Vero obtained from the Bank of cell cultures at the Institute of Cytology of the Russian Academy of Sciences.

Scheme of production experience.

Cells grown on medium RPMI-1640 with 10% serum fetal cow and placed in wells of 96-hole tablet was added to the virus at a final concentration of 102TED50/ml and the claimed compounds, dissolved in DMSO, final concentration of 100, 10 and 1 mg/l For each tested drug concentrations used 5 independent holes. Tablet incubated for 60 mi of the claimed compounds in the concentrations used.

The results were evaluated according to the presence cytopathogenic action of the virus on the cells after 36 hours of cultivation at 37oWith CO2the incubator.

In the experiment we used the following controls:

1. The control cell culture (ability to normal growth).

2. The virus control (ability to reproduce).

3. Control antiviral activity the antiviral drug acyclovir.

4. The control compounds (toxic compounds).

5. The control solvent (DMSO) toxicity.

To assess the cytopathic effect of the virus was calculated the number of unchanged cells in 100 fields formed a special grid eyepiece micrometer inverted microscope. The results obtained are presented in table. 3.

The obtained results indicate that given in table. 3 of the claimed compounds possess Antiherpes virus effect of activity comparable with that of standard drug acyclovir. The rest of the claimed compounds had less pronounced activity in suppressing the reproduction of the herpes virus in the selected experimental conditions.

Experiment 2. Determination of interferon-inducing activity savla is ulture human lymphocytes (data cells in the human body are the main producers of interferon). To obtain a culture of lymphocytes used fresh (12 hours after collection) blood of healthy donors (not the second group). For isolation of lymphocytes of heparinized blood obtained from a healthy donor were subjected to centrifugation in a density gradient ficoll-urografin 1.71 g/cm3to allocate fractions of immunocompetent cells. This fraction was taken and was diluted nutrient medium RPMI-1640 containing 5% serum fetal cow, 0.3 mg/ml L-glutamine, 100 u/ml penicillin, 50 mg/l streptomycin. The concentration of lymphocytes was considered after methylene blue staining and counting the number of cells in the chamber hemocytometer. The original solutions of the inventive substances were dissolved nutrient medium RPMT-1640 to a final concentration of substances was a number: 100 mg/l 10 mg/l 1 mg/l after making suspensions of lymphocytes. The final concentration of lymphocytes in the induction mixture was 3106cells/ml in Parallel with the experimental samples were stamped with the following controls:

1. Control of spontaneous production of interferons (IFN) lymphocytes.

2. Control of the process when exposed to a standardized inducer of IFN N-methyl-N-(a, D-glyukopiranozil)ammonium-10-metrocable is the fountain roller IFN - Neovir (sodium 10-methylanthracene-9-acridone) with relevant content DMC in the experimental samples.

4. Control of spontaneous production of interferon in the presence of DMCO in the number corresponding to the test samples.

Control and test samples were incubated 24 hours at 37oC. After incubation, the samples were tsentrifugirovanie at 2000 g for deposition of cellular elements and samples were selected IFN-containing supernatant, which were analyzed on a quantitative content of IFN. Sediment cells resuspendable in the same volume of nutrient medium were stained with the vital dye - Trifunovi blue and counted the number of cells in the chamber hemocytometer (as described above) to determine the cytotoxic effect of drugs. Quantitative determination of IFN in the control and experimental samples were produced using enzyme immunoassay system for IFN production LLP Protein contour Loop IF2 plus. To determine the amount of interferon in the sample used enzyme-linked immunosorbent method using horseradish peroxidase as indicator enzyme. The bound peroxidase activity was measured using an automatic photometer for Makelele activity of IFN standard solutions of IFN, containing a known amount of the drug. On the basis of the results obtained calibration curve was constructed, allowing the use of microprocessor automatic photometer to obtain data expressed in International Units of activity (IU). The results of the analysis are expressed in ME activity of IFN per ml in this induction system containing 3106lymphocytes/ml of Each experimental and control point was investigated in 4 Parallels.

Controls enzyme reactions:

1. Control DMCO with a nutrient medium.

2. Control system components (according to the manual).

All results were taken into account only in accordance controls the passport system.

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

As a result of the research showed that among the claimed compounds are samples, with the ability to induce the synthesis of IFN (table. 4).

The remaining compounds showed less activity.

Experiment 3. Determination of validity of the claimed achomatis D323 - the standard strains from the collection of Department of Microbiology, S.-Petersburg State medical University. AK. I. P. Pavlova. This strain was isolated from a patient with chlamydial urethritis is the morphology and physiological activity typical representatives of this species, sensitive to the action of drugs used for the treatment of chlamydial infection.

Used in the work cell culture Msso and L929 received from the Bank of cell cultures at the Institute of Cytology of the Russian Academy of Sciences.

Scheme of production experience.

Cells were grown in bottles of neutral glass in medium RPMI-1640 with the addition of 10% serum fetal cows. The experience was put in a glass (without toxicity) flat-bottomed bottles with cover glasses. The cells were made on Wednesday at a final concentration of 1106cells/ml After receipt of the monolayer in tubes made standard infecting dose chlamydia stored frozen at -70oC. Simultaneously to the cells was added compound at a final concentration of 100 mg/L. the Sample was centrifuged at 2400 g for 60 minutes at room temperature and incubated at 37oC for 2 hours. After that changed pittilo the s compounds in the same concentrations. In parallel, duplicate samples, using a medium without cycloheximide to prevent its influence on the investigated substance. Samples were incubated for 48 hours in CO2-incubator at 37oC.

Controls included:

1. The control cell culture.

2. The control action of solvents.

3. The control actions of chlamydia in the absence of any kind of drugs.

4. Control the sensitivity of chlamydia to standard antimicrobial drug ciprofloxacin.

5. The control of the tested compounds on toxicity to the cell cultures.

Evaluation of the results was performed by identifying chlamydial cytoplasmic inclusions using the method immunofluorescence (Msgothic Chlamydia trachomatis Direct Specimen Test) and chlamydial antigens using CylaMonoScreen (Russian-British Joint Venture 66 Regent's Academy Road London NW1 7SX) [6, 7]. The effect of the drug was determined by analyzing the state of the monolayer and the number of cells with CAI compared with the control (culture of cells infected with C. trachomatis D323) took into account the number of unchanged cells in 100 fields of view obtained when using an eyepiece micrometer.

The results of the control samples that satisfy the requirements of expellees;

control of growth of chlamydia in cell cultures is the presence of a CAI in the monolayer;

control standard antimicrobial drug reduction in CAI in the monolayer compared with the previous control;

control the toxicity of the inventive compounds - toxicity is missing;

the control action of solvents toxic effect on the cells is missing.

The results of the tests are presented in table. 5.

The obtained data show that the inventive compounds XI and XXV table. 5, have a pronounced activity against chlamydia, exceeding that of the standard drug ciprofloxacin.

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

Experiment 4. Determination of antimicrobial activity of the compounds.

To determine the antimicrobial activity was used a standard strain of Mycobacterium tuberculosis 37Rv, sensitive to all antimicrobial drugs. Assessment antimicrobacterial actions performed by the serial dilution method.

Compounds were dissolved in dimethyl sulfoxide (DMSO) and was titrated on Wed the concentrations of the drug in the environment of neighboring tubes differed in two times. The control used DMSO, which was titrated as a drug. The result was taken into account after 72 hours of culturing bacteria at 37oC.

M. tuberculosis H37Rv was grown on the environment Sotona containing 10% horse serum, and density of microbial suspensions at inoculation was 50106SOME.

As control were used known tuberculostatic drugs. The results obtained for the used strain, are given in table. 6.

Are given in table. 6 the data show that the tested compounds have antimicrobial activity in relation to the used strain of M. tuberculosis at a concentration of 100 mg/L.

The other claimed compounds showed less activity.

Experiment 5. Definition psychoparasite action of the claimed compounds.

In experiments on mice were evaluated by 10 parameters, indicating the possible development of depression behavior. Each parameter was evaluated in 2 points for each mouse with unchanged behaviour. The total score was 60 (2 x 10 x 3 mice). Reducing the number of scores below 40, 1 h after oral administration of the drug (300 mg/kg showed significant depression of conduct. In opressive action have not been tested.

Experiment 6. Evaluation of analgesic activity of the claimed compounds.

In a group of 3 mice was assessed by the time required for otdergivanija tail, placed under directional thermal radiation. The lengthening of the reaction time by more than 50% after intraperitoneal administration of the drug (30 mg/kg) indicated the presence of analgesic activity. As the comparison drug used analgin (2 mg/kg) (see table. 8). The rest of the claimed compounds for analgesic effect was not tested.

Experiment 7. Hepatoprotective effect of the claimed compounds.

Rats were injected subcutaneously with 50% solution of CCL4olive oil (1 ml/kg). The analyte was applied orally at a dose of 20 mg/kg 30 minutes before and after 7 hours after the introduction of the CCL4. A day in the blood of animals was determined by the activity of alanine aminotransferase (Alt) - a marker enzyme damage to the hepatic parenchyma. Reducing fermentee more than 30% compared with the control group was regarded as having hepatoprotective properties of the drug. As the comparison drug used vitamin E in a dose of 100 mg/kg (see table. 9). The other claimed compounds on hepatoprotector the

The test compound was administered orally using a stomach probe (300 mg/kg) or intraperitoneally (100 mg/kg) nonlinear white mice weighing 18-20 g (3 males and 3 females in each of the tested groups), and then observed their status within 72 hours. The absence of symptoms characteristic of toxic effects, and no animals died during a specified period of time allows to make a conclusion about the low toxicity of the studied compounds. In the presence of acute toxic effects the dose is reduced to identify the maximum tolerated dose [4] (see tab. 10).

The obtained results show that when taken through the mouth of the tested compounds at a concentration of 300 mg/kg not acutely toxic to mice.

The other claimed compounds on toxicity have not been tested.

Industrial applicability

The above examples and practical results of the synthesis and analysis of the claimed compounds confirm the possibility of laboratory and industrial synthesis of the claimed compounds means mastered the modern pharmaceutical industry, as well as their strict identification of common methods of control.

A series of experiment adut pronounced biological activities - antiviral against herpes simplex virus, interferon-inducing and protivodiareynoe, as well as antimicrobial, psychogenesis, analgesic and hepatoprotective effect.

These facts prove the achievement of the objectives of the invention: synthesized new compounds with low toxicity and a pronounced wide spectrum of biological actions.

Thus, in our opinion, the claimed substances meet all the requirements of the invention: they are new, non-obvious and industrially applicable.

Literature

1. Chatis, P. A., C. S. Crumpacker Resistance of herpesviruses to antiviral drugs. Antimicrob. Agents Chemother. 1992; 36: 1589-1595.

2. Pharmaceutical microbiology, Ed. by W. B. Hugo and A. D. Russel Blackwell Scientific Publications, Oxford, 1987, 511 p.

3. Esteban M. , ez E. Antiviral and antiproliferartive properties of interferons: mechanism of action. Prog. med. virol. 1985, 32: 159-173.

4. Irwin, S., Psychopharmacology, 1968, 13, p. 222-257.

5. Gentry, G. A., Lawrency N., Lushbaugh N. Isolation and differentiation of Herpes simplex virus and Trichomonas vaginalis in cell culture, J. of Clinical Microbiology 1985, Vol. 22. No. 2. P. 199-204.

6. Wang, S-P., J. T. Grayston Serotyping of Clamydia tracliomatis by inderect fluorescent-antibody staining of inclusions in cell culture with monoclonal antibodies. J. of Clinical Microbiology, 1991. Vol. 29. No. 7. P. 1295-1298.

7. Judson B. A., Lambert, P. P. Improved Syva MicroTrac Clamydia try. 1996, Vol. 147, No. 10-16.

9. Goldner, Dietz, Carstens // Ann. Chem, 1966, 691, p. 142; Ann. Chem., 1966, 698, R. 145, Ann. Chem., 1966, 699, p. 145.

10. Mashkovsky M. D. Medicines. In two parts. H. 1. - M.: Medicine, 1993, S. 550 prototype.

1. 2,4-Dioxo-5-arylidene-1,3-pyrimidines of General formula

< / BR>
where R is independently selected from the group: H, HE, lowest alkoxyl, halogen, nitro, di(lower)alkylamino;

n= 1-3, or two adjacent R along the benzene ring to which they are attached, for n=2,4, form a benzo, and dibenzo when n=2 form a 3,4-dioxolane ring.

2. Substance under item 1, wherein R=H, n=1.

3. Substance under item 1, characterized in that R=4-Cl, n=1.

4. Substance under item 1, characterized in that R=3-Cl, n=1.

5. Substance under item 1, wherein R=2-Cl, n=1.

6. Substance under item 1, characterized in that R=4-Br, n=1.

7. Substance under item 1, characterized in that R=4 HE, n=1.

8. Substance under item 1, characterized in that R=2,4-(OH)2n=2.

9. Substance under item 1, wherein R=2-OH, 5-Br, n=2.

10. Substance under item 1, wherein R=2-OH, 5-Cl, n=2.

11. Substance under item 1, characterized in that R=4-F, n=1.

12. Substance under item 1, characterized in that R=2,4-Cl2n=2.

15. Substance under item 1, characterized in that R=4-och3n=1.

16. Substance under item 1, characterized in that R=4-one, 3-och3n=2.

17. Substance under item 1, characterized in that R=4-NO2n=1.

18. Substance under item 1, characterized in that R=3-NO2n=1.

19. Substance under item 1, wherein R=2-NO2n=1.

20. Substance under item 1, wherein R=2-OH, 5-NO2n=2.

21. Substance under item 1, wherein R=2-HE, 5,6-benzo, n=3.

22. Substance under item 1, characterized in that R=3,4-dioxolane, n=2.

23. Substance under item 1, wherein R=2-NO2, 4,5-dioxolane, n=3.

24. Substance under item 1, characterized in that R=2,3,5,6-dibenzo, n=4.

25. Substance under item 1, wherein R=2-OH,3,5-J2n=3.

26. Substance under item 1, characterized in that R=4-N(CH3)2n=2.

 

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