RU2264396C2 - 6-alkyl-5-(2-isonicotinoylsulfohydrazoyl)uracil hydrochloride and pharmaceutical composition based on thereof - Google Patents

Abstract

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to new 6-alkyl-5-(2-isonicotinoylsulfohydrazoyl)uracil hydrochlorides of the general formula (I)

wherein R means alkyl comprising 1-4 carbon atom. Compounds possess an anti-mycobacterial and an immunotropic activity and can be used as an immunomodulating agent and an anti-mycobacterial agents. Also, invention relates to a pharmaceutical composition based on thereof.

EFFECT: valuable medicinal properties of compounds.

4 cl, 10 tbl, 2 ex

Description

The invention relates to the field of biologically active substances, in particular to 6-alkyl-5- (2-isonicotinoylsulfohydrazoyl) uracil hydrochlorides, which have antimycobacterial and immunotropic activity and can be used as immunomodulator and antimycobacterial agents, as well as to pharmaceutical compositions based on them.

Known alkaline salts of N- (6-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfon) -N'-isonicotinoyl hydrazide of the formula (A) (Isodinafon)

where R is alkyl with 1-4 carbon atoms and M is an alkali metal having similar activity with toxicity of 7000 mg / kg (see international patent application WO 01/32650). However, their activity is not high enough (see the following tables).

Also known is N- (6-methyl-2.4-dioxo-1.2.3.4-tetrahydro-5-pyrimidinesulfone) -N-isonicotinoyl hydrazide hydrate (B) having antimycobacterial and immunomodulating activities (patent RU 2141322). However, this compound (B) is not soluble in water, saline and other solvents, has poor solubility in biological fluids, which indicates its low biological activity, low bioavailability.

The basis of the invention is the task of finding new structurally similar compounds with higher activity.

The problem is solved by the proposed 6-alkyl-5- (2-isonicotinoylsulfohydrazoyl) uracil hydrochlorides of the general formula (I)

where R is alkyl with 1-4 carbon atoms.

6-Methyl-5- (2-isonicotinoylsulfohydrazoyl) uracil hydrochloride of the formula (I) is a white to cream crystalline powder with a melting point Tm = 253-254 ° C.

The proposed compounds are prepared by reacting isonicotinic acid hydrazide with 6-methyluracil-5-sulfochloride in a solvent.

The compounds of formula (I) have antimycobacterial and immunotropic activity.

The problem is also solved by the proposed pharmaceutical composition having antimycobacterial and immunotropic activity, including an active substance and target additives, the difference of which is that it contains 6-alkyl-5- (2-isonicotinoylsulfohydrazoyl) uracil hydrochloride of the general formula (I) as an active substance in an effective amount.

The dosage depends on the age, condition and weight of the patient, as well as on the type of administration. According to the invention, the proposed pharmaceutical composition contains an active substance in an amount of from 0.1 to 4 g.

The toxicity of the proposed compounds was determined on outbred white mice weighing 18-20 g in an acute experiment using the Litchfield-Wilcoxon method and amounted to more than 7000 mg / kg

The claimed compounds were tested in animal experiments.

Determination of the immunotropic activity of the proposed hydrochloride

a) The effect on humoral immunity was studied using the method of local hemolysis in the gel. Mice of F1 hybrids (CBA × C ₅₇ BI ₆ ) were immunized with ram erythrocytes at a dose of 5 × 10 ⁶ and immediately after that the studied substances were administered both orally in a starch suspension and parenterally. On the 5th day, the number of antibody-forming cells (AOKs) in the spleen of mice was counted and the immune response stimulation index was determined in relation to the number of AOKs in the experimental group and the number of AOKs in the control group.

The data are presented in table 1.

Table 1.
The effect of the studied compounds on the accumulation of antibody-forming cells (AOKs) in the spleen of mice (the difference is significant (p less than 0.01) in relation to the norm). Drug name Route of administration Dose m kg / mouse The number of AOK in the spleen Number of animals Immune Response Stimulation Index The control orally 0.5 ml starch. suspensions 500 ± 50 12 1,0 Hydrochloride orally 500 2850 ± 150 12 5.3 ± 0.5 Isodinafon orally 500 2360 ± 250 12 4.6 ± 0.7 The control parenteral 0.2 ml of fiziol. solution 510 ± 50 12 1,0 Hydrochloride parenteral 100 1800 ± 100 12 403 ± 0.6 Isodinafon parenteral 100 1650 ± 280 12 3.20 ± 0.6

Indices of stimulation of the immune response of hydrochloride and isodinafon with the introduction of drugs inside are significantly indistinguishable. This suggests that the studied drugs have a similar effect on humoral immunity, tested in the reaction of accumulation of AOK in the spleen of mice.

b) The effect of the studied drugs on the proliferation of T-lymphocytes was studied in an in vitro model. For this, a preliminary 3-hour incubation of human mononuclear cells was carried out in the presence of different concentrations of each test substance. We studied a wide range of concentrations of each test substance: from 0.01 to 5 μg / L. As a negative control, saline 0.9% NaCI was added.

The results of the influence of new compounds on the intensity of proliferation of human T-lymphocytes in cell culture in vitro are presented in table 2.

Table 2.
The effect of hydrochloride on the proliferation of T-lymphocytes in an in vitro culture. A drug Dose, mcg / ml The intensity of the inclusion of 3H-thymidine DNA of cultured cells (pulse / min) Spontaneous Activated PHA Saline 800 20002 Hydrochloride 5 900 20132 1 1275 25,400 0.1 1301 38000 0,03 1980 31150 0.01 1530 29990 Isodinafon 5 620 19142 1 812 22068 0.1 1144 28145 0,03 1762 34284 0.01 1496 29586

Thus, the proposed hydrochloride as well as isodinaphone has both its own mitogenic activity and in response to phytohemagglutinin, but exhibits this activity when added in a lower dose.

c) The influence of the studied drugs on the phagocytic activity of macrophages was studied by carcass clearance in blood taken from the retroorbital sinus of mice that received oral preparations. The results were evaluated by the phagocytic index (table 3).

Table 3.
The influence of the studied drugs on the phagocytic activity of macrophages (the difference is significant (p less than 0.01) in relation to the norm). A drug Route of administration Dose, mcg / mouse Phagocytic index Number of animals Starch suspension orally 0.5 ml 4.2 ± 0.06 12 Hydrochloride orally 500 7.1 ± 0.03 12 Isodinafon orally 500 6.64 ± 0.12 12 Saline parenteral. 0.2 ml 4.1 ± 0.07 12 Hydrochloride parenteral. 100 7.5 ± 0.06 12 Isodinafon parenteral. 100 7.12 ± 0.09 12

The results shown in the table indicate that the hydrochloride stimulates the phagocytic activity of macrophages to a greater extent than isodinafon with any method of administration.

d) The immunocorrecting ability of the hydrochloride was determined on the model of immunodeficiency caused by irradiation of F ₁ mice (CBA x C ₅₇ BI ₆ ) at a dose of 4 Gray.

It was shown that irradiation of mice with a dose of 4 Gray reduces the immune response by 15-20 times, its recovery begins on day 6-8 and ends by 30 days (Yarilin A.A., Polushkina E.F., Filatov P.P. ionizing radiation on the ratio of lymphoid cell populations in mice, Radiobiology, 1976, v. 16, No. 3, pp. 451-454). When using this model, test preparations were administered test animals at doses of 500 μg / mouse orally in 0.5 ml of starch suspension and 100 μg / mouse parenterally in 0.2 ml of saline. The drugs were administered three times on 6.7.8 days after irradiation. The control group of animals received 0.5 ml of starch suspension or 0.2 ml of saline. On the 15th day, the amount of AOK in the spleen was determined in these animals by local hemolysis in gel.

The data obtained are presented in table 4.

Table 4.
The effect of hydrochloride on the restoration of the formation of antibody-forming spleen cells in irradiated animals. A drug Route of administration Dose mcg / mouse Number of animals AOK on the spleen Stimulation index Irradiated control 12 309 ± 36 Hydrochloride Orally 500 12 1080 ± 39 3.5 ± 0.4 Isodinafon Orally 500 12 995 ± 12 3.2 ± 0.5 Physical control solution Parenteral 0.2 ml 12 312 ± 40 Hydrochloride Parenteral 100 12 1200 ± 54 4.0 ± 0.2 Isodinafon Parenteral 100 12 1074 ± 12 8 3.5 ± 0.5

Based on the obtained data on the restoration of the immune response after irradiation, we can conclude that the hydrochloride exhibits a greater immunocorrective activity than in comparison drugs.

Determination of the anti-tuberculosis activity of hydrochloride.

Activity was tested in comparison with isodinafon. For infection, laboratory strains of Mycobacterium tuberculosis N ₃₇ RV normal and tubazidoustable up to 100 mg were used. 230 CBA mice were infected intravenously with 0.25 mg of tuberculosis microbial culture in 0.5 ml of saline. The experiment lasted 2 months. Under observation were 20 groups of animals, 15 mice in each group, all animals were kept in the same vivarium conditions on a standard diet. The control groups received throughout the experiment through a probe 5 times a week 0.5 ml of starch suspension or 0.2 ml of parenteral saline. Experimental groups of animals received oral preparations at various doses in 0.5 ml of starch suspension or in 0.2 ml of parenteral saline. All animals, both dead and slaughtered, were weighed at the end of the experiment, their internal organs were also weighed, the degree of damage to the internal organs was determined, assessed as "+", in addition, the effect of the drug was judged by the average life expectancy (LSS).

The experimental results are shown in table 5.

Table 5.
The effect of the test substances on anti-tuberculosis activity. Drugs, doses, route of administration H ₃₇ RV H ₃₇ RV-tubazide resistance SPG, day Lung weight, mg Lesion index SPG, day Lung weight, mg Lesion index Control 0.5 ml collapse. 24.2 619.2 2.48 23.8 632.4 2.42 oral suspension ± 2.7 ± 50.4 ± 0.22 ± 0.22 ± 70.2 ± 0.19 Hydrochloride 100 3,5 500 ± 105 37.3 450.5 1.7 mcg / mouse orally ± 5.0 51,2 ± 0.13 ± 3.8 ± 51.7 ± 0.2 Isodinafon 100 33,4 526.1 2.01 32,4 492.8 1.96 mcg / mouse orally ± 5.1 ± 47.2 ± 0.12 ± 3,5 ± 63.4 ± 0.21 Control 0.2 ml 22.8 628.3 2,52 24.1 648.2 2.72 saline parenteral ± 2.2 ± 61.4 ± 0.21 ± 4.0 ± 71.4 ± 0.21 Hydrochloride 31,2 530 1.7 33,4 590.3 1.9 50 mcg / mouse parenterally ± 2.1 ± 21.5 ± 0.03 ± 3.2 ± 75.5 ± 0.1 Isodinafon 30.5 580.2 ± 1.80 31.6 607.8 2.03 50 mcg / mouse parenterally ± 4.6 46.2 ± 0.09 * ± 3.1 ± 59.2 ± 0.21 * * Values significantly different from the control, P <0.05

The above results show that hydrochloride has a pronounced anti-tuberculosis effect both on mycobacteria sensitive to most anti-TB drugs and on mycobacteria resistant to tubazide. Judging by the index of lung damage in infected animals, the anti-tuberculosis activity of hydrochloride is manifested to a greater extent than in comparison drugs.

Determination of anti-leprosy activity of hydrochloride in comparison with isodinafon.

The anti-leprosy activity of hydrochloride in comparison with isodinafon was studied in mice of CBA males infected with the laboratory strain of Kur-4 mycobacterium leprosy, taken in an amount of 5000 and introduced intraplantally according to the Shepard method. The experiment lasted 6 months. Under observation were 5 groups of 20 mice in each group. All mice were kept under identical vivarium conditions on a standard diet.

The first group is the control: for 6 months, mice of this group received 0.5 ml of starch suspension through a probe 5 times a week. In another series of experiments, 0.2 ml of physiological saline was administered parenterally to the control animals at the study time, and experimental animals were diluted with saline.

After 6 months, the animals were clogged with cervical displacement of the spine, organs were subjected to bacterioscopic examination, no pathological abnormalities characteristic of any group were found. During bacterioscopic examination of the site of infection (paw), they were ground in 2 ml of a 0.1% albumin solution, a smear was made from 0.01 ml of the suspension, stained with Ziehl-Nielsen and the number of mycobacteria per 1 mouse in each group was counted.

The research results are presented in table 6.

Table 6.
The effect of the test substances on anti-leprosy activity (the difference is significant (p less than 0.01) in relation to the norm). Group Μg / mouse dose The number of mice in the group Number of mycobacteria in the foot x10 ⁶ % infected animals The control 0.5 ml crash. susp. peror. twenty 1.14 ± 0.12 100 Hydrochloride fifty twenty - 0 Isodinafon fifty twenty - 0 The control 0.2 ml parenteral saline twenty 1.27 ± 0.14 100 Hydrochloride 25 twenty - 0 Isodinafon 25 twenty - 0

As follows from the above data, the hydrochloride in all the studied doses and methods of use exhibits pronounced antileprosy activity as well as isodinafon at a dose of 25 μg / mouse.

The study of the antimicrobial action of hydrochloride

The antimicrobial effect of hydrochloride in comparison with isodinafon was studied on cultures of Bacillus cereus and Staphylococcus aureus. To determine the antimicrobial effect of the preparations on B. cereus, 100 mg of each preparation was taken, diluted in 10 ml of phosphate buffer (pH 7.0), and 1 ml was added to test tubes containing 1 ml of molten medium No. 1 (USSR State Pharmacopoeia, ed. e XI, issue 1, volume 2, p. 200) and 1 ml of a daily broth culture of B. cereus diluted 1000 times with saline. The contents of the tube were immediately poured onto a Petri dish containing 15 ml of frozen medium No. 1, the plates were placed in a thermostat at a temperature of 30 to 35 ° C.

To determine the antimicrobial effect of drugs on St. aureus took 100 mg of each drug, was diluted in medium No. 8 (State Pharmacopoeia of the USSR, ed. XI, issue 1, v.2, p. 208) and 1 ml was added to tubes containing 4 ml of molten medium No. 10 (State Pharmacopoeia of the USSR, ed. XI, issue 1, v.2, p. 208) and 1 ml of daily broth culture Staphylococcus aureus, diluted with saline 1000 times. The contents of the cork were immediately poured into Petri dishes containing 15 ml of frozen medium No. 10, the plates were placed in a thermostat with a temperature of 30 to 35 ° C. The experiment lasted 5 days.

In the absence of growth of test strains on the corresponding nutrient media, the antimicrobial effect of the drugs is noted. The results of the experiment are presented in table 7.

Table 7.
The antimicrobial effect of hydrochloride in comparison with isodiaphone. Test culture Hydrochloride Isodinafon 24 hours 48h 5 days 24 hours 48h 5 days S. cereus - - - - - - St. aureus - - - - - ±

+ - the presence of microbial growth

- - lack of growth of microorganisms

± - slight growth of microorganisms

As the results of the experiments show, hydrochloride has a bactericidal effect on microorganisms of B. cereus and a pronounced bacteriostatic effect on St. aureus.

The method of obtaining the proposed compounds of formula (I) is illustrated by the following example.

Example

1.26 g (0.01 mol) of isonicotinic acid hydrazide and 2.245 g (0.01 mol) of 6-methyluracil-5-sulfonyl chloride in acetonitrile are mixed at the boiling point of the solvent for 5 hours.

The precipitated hydrochloride is filtered and purified in several stages as follows: 0.5 g of hydrochloride is boiled in 15 ml of alcohol for 10 minutes, then filtered, the precipitate is washed with 6 ml of alcohol, and dried in air. This is repeated one or two more times until only trace amounts of impurities remain.

3.115 g of 6-methyl-5- (2-isonicotinoylsulfohydrazoyl) uracil hydrochloride are obtained (approximately 90% yield), which is a white to cream crystalline powder. _Mp = 253-254 ° C

Formulation Examples of Pharmaceutical Compositions

Capsules

Hydrochloride in gelatin capsules of 0.1 g and 0.2 g is obtained by packing manually in capsules No. 1 and No. 0 using capsules with a locking device. Composition: hydrochloride - 0.1 g and 0.2 g without fillers and preservatives.

A portion of the hydrochloride 10.0 g is placed on the capsule plate, distributed on the surface of the plate and then in accordance with the description of the capsule process.

Gelatin capsules consist of: quinoline epu - 0.05%, titanium dioxide - 1.0%, gelatin - up to 100%. The substances included in the capsule are approved for medical use in Russia.

Tablets

The active substance is a compound of formula (I) in an amount of 100 mg, 200 mg - mass fraction of 40%. Targeted additives (lactose, aerosil, calcium stearate) - mass fraction of 60%.

The main and auxiliary substances are mixed in the ratios indicated in table 8 (calculated on the weight of the tablet):

Table 8 The mass of tablets (M _t ), g Mass composition,% 0.250 0,500 Hydrochloride - 0.1 hydrochloride - 0.2 40 Lactose - 0.144 Lactose - 0.288 57.6 Aerosil - 0.004 Aerosil - 0.008 1,6 Stearate Ca - 0.002 Stearate Ca - 0.004 0.8

The diameter of the balls is 5 mm. The mass of balls per 100g of the finished form is 500 g.

The pressing of the tablets is carried out on an RTM-12 press. The tablets are coated on the basis of acetylphthalyl cellulose (AFC) on a Strea-1 apparatus. Coating composition (mass parts): acetone - 56.4; ethyl alcohol 96% - 37.6; castor oil - 1.0; AFC - 5.0; tropeolin - add to the light brown color of the solution. Coating consumption: amount of coating (ml) = 50% by weight of tablets (g).

Solution for injection

Hydrochloride for injection is a powder of a medicinal product, including the active substance and its pharmaceutically acceptable salts in a dosage of 0.1-0.5 g.

Specific formulations are shown in table 9.

Table 9 Components Amount ml 1 2 3 compound of formula (I), g 0.1 0.2 0.5 Solvent Acceptable Solvent 5 ^- Acceptable Solvent ^- 5 Acceptable Solvent 5

An experiment on animals (rabbits) showed that about 50% of this compound is found unchanged in the products of their vital activity. Biological tests of hydrochloride (I) showed that its effect on humoral immunity when administered orally is slightly higher than that of compound (B), whereas with parenteral administration the same effect is achieved when 8 times lower doses of hydrochloride are administered.

The hydrochloride compared with the above compound (B) showed antimicrobial activity, in the case of B. cereus - bactericidal, and in the case of St. aureus - bacteriostatic, as evidenced by the data in table 10.

Claims (3)

1. 6-Alkyl-5- (2-isonicotinoylsulfohydrazoyl) uracil hydrochloride of the general formula (I)

where R is alkyl with 1-4 carbon atoms. 2. The hydrochloride salt according to claim 1, exhibiting antimycobacterial and immunotropic activity. 3. A pharmaceutical composition having antimycobacterial and immunotropic activity, comprising an active substance and target additives, characterized in that it contains 6-alkyl-5- (2-isonicotinoylsulfohydrazoyl) uracil hydrochloride of the general formula (I) according to claim 1 in claim 1 as an active substance effective amount.