UA72286C2 - Alkaline salts n-(6-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinsulfone)-n'-izonicotinoylhydrazide which can be used as antimicrobial and immunotropic agents, a method for the preparation thereof, use thereof as immunomodulator and pharmaceutical composition based thereon - Google Patents

Abstract

Alkaline salts of N-(6-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone)-N'-isonicotinoylhydrazide of formula (I), where R is 1-4 carbon atoms alkyl and M is alkali metal, which can possess antimicrobial and immunothrope activities and can be used as immunomodulator are offered, as well as the method for producing these salts and pharmaceutical composition based on them. The offered salts may be used in treating chronic recurring diseases complicated with bacterial infections on the immunodeficiency background, also they may be used as antimicrobial means. (I)

Description

The invention relates to the field of biologically active substances, in particular, alkaline salts of M-(b-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone)-M'-isonicotinoylhydrazide, which can be used as an antimicrobial - bny and immunotropic agents and can also be used as an immunomodulator, as well as a method of their preparation and a pharmaceutical composition based on them.

Diucifone is known to have immunomodulatory properties and to exert an immunostimulating effect on humoral and cellular immunity (USSR copyright Mo 459228), but it does not have its own mitogenic activity, but is able to increase the proliferation of T cells only in response to phytohemagglutinin (FGA ). The toxicity of diucifon is 2600 mg/kg.

Also known is M-(b-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone)-M'-isonicotinoyl hydrazide hydrate (isophone) (RF patent Mo 2141322), which has antimicrobial and antimycobacterial properties, however, due to its poor solubility in water, it has low biological activity, which is confirmed by an experiment on animals (rabbits), in which it is shown that about 5095 isophones are found unchanged in waste products.

The invention is based on the task of creating new substances for use as antimicrobial and immunotropic agents, developing a method of obtaining these compounds, using them as an immunomodulator, and creating a new pharmaceutical composition based on them.

The given task is solved by the proposed alkaline salts of M-(6b-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone)-M'-isonicotinous hydrazide of formula I:

AND

80,-K-M-s- nt More

ХК мм M

N -- shk

M where B is alkyl with 1-4 carbon atoms and M is an alkali metal.

The compounds of formula I have antimicrobial and immunotropic activity and can be used as an immunomodulator containing the active substance in an effective amount in chronic recurrent diseases complicated by bacterial infections against the background of immunodeficiency.

The problem is also solved by the method of obtaining compounds of the formula I, which consists in mixing the derivatives of the isophone of the general formula I

NM | 5О, -МНА-А-МН-с-о0 ! in Kk with

N | r no

M where A has the above value, with the appropriate alkali, and the resulting salt is precipitated with a water-miscible solvent.

The problem is also solved by the claimed pharmaceutical composition, which contains as an active substance alkaline salts of the general formula I in an effective amount and additives.

The claimed compound of formula II, where M is Ma and A is methyl (isodinaphon), is a white amorphous powder with m.p. 300"C, very soluble in water.

The toxicity of isodinafon was determined on white purebred mice weighing 18-20 g in an acute test by the Litchfield-Wilcoxon method and was more than 7000 mg/kg. etc

The claimed compound was tested in experiments on animals.

Determination of the immunotropic activity of isodinafon. a) The effect on humoral immunity was studied using the method of local hemolysis in a gel. Mice of E1 hybrids (SBA x C57Vivie) were immunized with ram erythrocytes in a dose of 5x10 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 (AUC) in the spleen of mice was counted and the index of stimulation of the immune response was determined by the ratio of the number of AUC in the study group to the number of AUC in the control group.

The data are shown in Table 1.

Table 1

The effect of the studied compounds on the accumulation of antibody-forming cells (AUC) in the spleen of mice

Index of style - R

Name of the preparation - Method of administration Dose Amount of AUC in | The number of mulation of immu- probability of rat μg/mouse in the spleen of different animals relative to control control

11117771, suspension | HER! solutions

The indices of stimulation of the immune response of isofon, soluble diucifon and isodinafon when the drugs are administered inside are probably indistinguishable from each other. This suggests that the studied drugs have the same effect on humoral immunity, which is tested in the reaction of the accumulation of AHAUK in the spleen of mice.

The proposed drug isodinafon also works with parenteral administration in a smaller dose, which allows saving the drug. It acts to the same extent as soluble diusifon, but the advantage of isodinafon is its lower toxicity compared to soluble diusifon both when administered orally and when administered parenterally (the LD of isodinafon when administered orally is 700 ug/kg, and of soluble diusiphon 2600mg/kg (2136-3042); with parenteral administration of LdDBzo, isodinaphon is 1750mg/kg (1286-2380), and diucifon is 110Omg/kg (887--1364)). b) The effect of the studied drugs on the proliferation of T-lymphocytes was studied on the model of ip migo. To do this, a preliminary 3-hour incubation of mononuclear cells and a person was carried out in the presence of different concentrations of each substance under study. A wide range of concentrations of each investigated substance was studied: from 0.01 to 5 μg/l. A physiological solution of 0.990 Mass was added as a negative control.

The results of the effect of new compounds on the intensity of proliferation of human T-lymphocytes in the culture of IP myo cells are shown in Table 2.

Table 2

The effect of isodinafon on the proliferation of T-lymphocytes in the culture of the ip migo of a deer Gen nove yasnnnn

Drug Dose, μg/ml and cells (pulse/min) 11111111 Spontaneous./ | Activated FGAZ 800 20002 871 20120

Physiological solution isofon! 1272 25455 0.1 1818 37890 0.03 1152 24736 0.01 901 19995 5 800 20150 1 799 26400

Diucifone cal. 0.1 804 28946 0.03 850 24214 0.01 860 22811 5 620 19142 1 812 22068

Isodinafon 0.1 1144 28145 0.03 1762 34284 0.01 1496 29586

Thus, the proposed drug, like isofon, has both its own mitogenic activity and in response to phytohemagglutinin, but exhibits this activity when added in a smaller dose. c) The effect of the studied drugs on the phagocytic activity of macrophages was studied by the clearance of the carcass in the blood taken from the retroorbital sinus of mice that received the drugs orally. The results were evaluated according to the phagocytic index (table 3). μg/mouse dex control animals

Hepelnnsyu|entyu | on 1 She 0121 10095 o

41 0.07 12 11111111

Isofon parenteral. 100 4.6250.08 12 -0.01 11095 o

Isodinafon parenteral. 100 7. 1250.09 12 -0.01 16995

The results presented in the table indicate that isodinafon stimulates the phagocytic activity of macrophages as well as soluble diucifone by any route of administration, but to a greater extent when administered orally, while isofon acts only when administered orally. d) The immunocorrective ability of isodinafon was determined on the model of immunodeficiency caused by irradiation of PE) (SVAxC57VIb) mice at a dose of 4 Gray. It has been shown that irradiation of mice at a dose of 4 Gray reduces the immune response by 15-20 times, its recovery begins on 6-8 days and ends up to 30 days (Yarilyn A.A., Polushkina Z3.F., Filatov P. .P. Effect of ionizing radiation on the ratio of populations of lymphoid cells in muscles, Radiobiology, 1976, vol. 16, Mo. 3, pp. 451-454). When using this model, experimental animals were administered tested drugs in doses of 500 μg/mouse orally in 0.5 ml of starch suspension and 100 μg/mouse parenterally in 0.2 ml of physiological solution. The drugs were administered three times on the 6th, 7th, and 8th day after irradiation. The control group of animals received 0.5 ml of starch suspension or 0.2 ml of physiological solution. On the 15th day, the amount of AUC in the spleen of these animals was determined by the method of local hemolysis in a gel.

The obtained data are shown in Table 4.

Table 4

The effect of isodinafon on the restoration of the process of formation of antibody-forming cells of the spleen in irradiated animals.

Doses Amount of AUC per se- | Stim index - | most likely

Drug Method of administration | µg/"mice and other animals of the ladder of lation compared to the control

Rossini PNYA NN NS NI VAYE: Z NOYA POLON troll 1013-26 3,610.5

Diucifone cal. 780295 2.5:0.8 995412 3.240.5 4185252 1.4850.3

Diucifone cal. 8102116 2.150, 10745128 3.520.5

On the basis of the obtained data on the restoration of the immune response after irradiation, it can be concluded that isodinafon exhibits an immunocorrective activity that is not inferior to the comparison drugs when administered orally and is 2 times superior to isofon when administered parenterally in a dose that is 5 times smaller.

Determination of the antituberculosis activity of isodinafon.

The activity was checked in comparison with isofon and a highly active immunomodulator, soluble diucifon.

For infection, laboratory strains were used: mycobacterium tuberculosis Nz7 VM ordinary and tubazid-resistant to 100 mg. 230 mice of the SBA line were infected intravenously with 0.25 mg of tuberculosis microbacteria culture in 0.5 ml of physiological solution. The test continued for 2 months from September 10 to November 12, 1996. 16 groups of animals were observed, 15 mice in each group, all animals were kept in the same vivarium conditions on a standard diet. Control groups received 0.5 ml of starch suspension or 0.2 ml of saline parenterally through a tube 5 times a week throughout the experiment. The studied groups of animals received drugs orally in different doses in 0.5 ml of starch suspension or parenterally in 0.2 ml of physiological solution. 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 internal organs was determined, which was evaluated in "w", in addition, the effect of the drug was judged by the average life span (LSL).

The results of the experiments are shown in Table 5.

Table 5

The effect of the studied substances on antituberculosis activity

On; VM - tubazid resistance

Drugs, doses, sup- Lung weight Ura index Lung weight Ura index

MG suspension MG suspension orally mcg/mouse orally

Isophon 100 mcg/mouse mcg/mouse orally

Control 0.2 ml of parenteral saline solution - 22.8х2.2 | 628.3361.4 2.5250.21 24.154.0 64825714 2.120 21 flax

Diucifone cal. 50 μg/mouse parentera- | 26.252.1 612.4552.4 2.350.22 26.7521 637.0572.1 2.40-0.31 flax parenterally

Isodinafon 50 μg/mouse parente- 30.5:4.6 | 580.25346.2 1.8020.097 Z31.6х3.1 607.8259.2 2.0350.217 ralno "Values that probably differ from the control, P"e0.05

As the obtained results show, isodinafon has a pronounced antituberculosis effect both on mycobacteria sensitive to most antituberculosis drugs and on mycobacteria resistant to tubazid. Judging by the lung damage index of infected animals treated with isodinafon and diucsifon soluble, the antituberculosis activity of isodinafon is revealed by parenteral administration to a greater extent than in comparison drugs.

Determination of anti-leprosy activity of isodinafon in comparison with isofon and soluble diucifon.

The anti-leprosy activity of isodinafon compared to soluble isofon and diucifon was studied in male mice of the SBA line, infected with the laboratory strain of Mycobacterium Leprosy Chicken-4, taken in the amount of 5000 and injected intraplantarly according to Shepard's method. The experiment would continue for months from February 7 to August 4, 1996. We observed 5 groups, 20 mice in each group. All mice were kept in the same vivarium conditions on a standard diet.

The first group is a control group: for 6 months, mice in this group received gavage 5 times a week

About 5 ml of starch suspension. In another series of experiments, control animals were parenterally injected with 0.2 ml of physiological solution into the test tubes, and the subjects - drugs diluted in physiological solution.

After two months, the animals were killed by cervical spine displacement, the organs were subjected to bacterioscopic examination, no pathological deviations characteristic of any group were found. During bacterioscopic examination, the site of infection (paw) was rubbed in 2 ml of 0.195 albumin solution, a smear was made from 0.01 ml of the suspension, stained according to Ziel-Nielsen, and the number of mycobacteria per 1 mouse in each group was counted.

The results of the research are shown in Table 6.

Table 6

The effect of the studied substances on the anti-leprosy activity of "shey" in the group of swarms in the foot of 6 animals trolled

Diucifon solutions 50 | 20 | 0048:30007 | 60 | "001 "( pzofon.//// | 7777/5077 777720 | 777-777 77171717171077117 Fr "0010 bzodilaphoni | -:(/ 50 7 | 77/20 | 7777-1177 "0010 tyut | in sem | 500 parenterally

Ozodynchafonyi | -:(: 25 | 20 | yuyu - | ..yuyuyuyuTo0 5 Byuzhk | «001 (

As follows from the given data, isodinafon in all studied doses and methods of application shows pronounced anti-leprosy activity, in contrast to soluble diucifone and isofon, which do not have anti-leprosy activity at a dose of 25 μg/mouse.

Study of the antimicrobial action of isodinafon.

The antimicrobial effect of isodinaphon compared to soluble diucsiphon and isophon was studied on cultures of Vasii segeiz and Biarnuiosossiv aigeiv. To determine the antimicrobial effect of drugs on B, segeiv5, 100 mg of each drug was taken, diluted in 10 ml of phosphate buffer (pH 7.0) and 1 ml was added to test tubes containing this ml of molten medium Me 1 (State Pharmacopoeia of the USSR, ed. XI, vpp. 1, vol. 2, p. 200) and 1 ml of daily broth culture of V. segeivs, diluted 1000 times with physiological solution. The contents of the test tube were immediately poured into a Petri dish containing 15 ml of solidified Me 1 medium, the dishes were placed in a thermostat at a temperature from 30 to 3570.

To determine the antimicrobial effect of drugs on 51. ashgeiz, 100 mg of each drug was taken, diluted in Me 8 medium (State Pharmacopoeia of the USSR, ed. Kh, vp. 1, vol. 2, p. 208) and added to 1 ml in test tubes, containing 4 ml of molten medium Me 10 (State Pharmacopoeia of the USSR, ed. X1, vol. 1, vol. 2, p. 208) and 1 ml of daily broth culture of biarpuiosossisis ashgeiv, diluted 1000 times with saline.

The contents of the test tube were immediately poured into Petri dishes containing 15 ml of solidified Mo 10 medium, the dishes were placed in a thermostat with a temperature from 30°C to 35°C. The experiment lasted 5 days.

In case of no growth of the test strains on the appropriate nutrient media, the antimicrobial effect of the drugs is ascertained. The results of the experiment are shown in Table 7.

Table 7

Antimicrobial action of isodinaphon in comparison with diusiphon soluble and isophon

All | x | «| 5 | x | 5 | - |:

Indeed/ | «| ya | 5 | | "x || - | - | x -- presence of growth of microorganisms - absence of growth of microorganisms y - insignificant growth of microorganisms

As the results of the conducted experiments show, isodinafon exerts a bactericidal effect on the microorganisms of V. segei5 and has a pronounced bacteriostatic effect on 51. ashgeiv na. in contrast to isofon, which does not have an antimicrobial effect, and from soluble diucifon, which has a weak bacteriostatic effect.

Other alkali salts of formula I show similar activity.

The method of obtaining compounds of formula I is illustrated by example 1.

Example 1.

Disodium salt of M-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone)-M'-isonicotinoylhydrazide. 2 g (6.2 mmol) of isofone is dissolved in 1 mL of a solution containing 0.52 g (1 mL) of caustic soda. The obtained slightly yellowish solution is filtered through a glass filter and the filtrate is transferred to a three-necked flask. Cool to room temperature and, with intensive stirring, add a boml of ethanol. Stir for 2 hours until the salt completely settles. The precipitate is filtered, washed with 20 ml of alcohol, dried in an oven at 5076.

Yield 1.7g (74.2905). White amorphous powder, m.p. 300"C, very soluble in water.

Examples of formulations of pharmaceutical compositions are given below.

Injections: active substance isodinafon - 200 mg solvent - novocaine 0.595 or 0.2595 - zhml Capsules: active substance isodinafon - 100 mg, 200 mg, 500 mg.

Tablets: active substance isodinafon - 200 mg fillers: starch - ZOmg talc - 10 mg lactose - 10 mg

Ointment: active substance isodinafon - 2g base: emulsion wax - 20g petroleum jelly - 20g glycerin - 20g distilled water - 8g

Thus, the proposed alkaline salts of the formula | show a wide spectrum of immunotropic activity in lower doses than isofon; retain anti-leprosy and anti-tuberculosis effects and act on pathogenic organisms. In this regard, the claimed compounds can be used in medicine as an immunomodulator in the treatment of chronic recurrent diseases complicated by bacterial infections against the background of immunodeficiency, as well as as antimicrobial agents.

Claims (5)

1. Alkaline salts of M-(6b-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone)-M'-isonicotinoylhydrazide of the general formula I: ГИ и 5О,-М-М-С - 2 -th nyu Y M Z y: om shk: H r M where A is an alkyl with 1-4 carbon atoms and M is an alkali metal. 2. Alkaline salts of M-(6b-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone)-M'-isonicotinoylhydrazide of the general formula I according to claim 1 for use as antimicrobial and immunotropic agents. 3. The method of obtaining alkaline salts of M-(6-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone)-M'-isonicotinoyl hydrazide of the general formula | according to claim 1, which consists in the fact that the isophone derivatives of the general formula HER are mixed) 50, -МН--МН -с-0 НМ | 2 o in | with Ko) N r ? M ШЧИ where K has the above value, with an appropriate alkali, and the resulting salt is precipitated with a water-miscible solvent. 4. A pharmaceutical composition containing an active substance and additives, which differs in that it contains the alkaline salt of M-(6-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone) as an active substance )-M'- isonicotinoyl hydrazide of the general formula I according to claim 1 in an effective amount. 5. The use of alkaline salts according to claim 1 as an immunomodulator containing an active substance in an effective amount, in chronic recurrent diseases complicated by bacterial infections against the background of immunodeficiency.