WO2001032650A2

WO2001032650A2 - Alkaline salts of n-(6-alkyl-2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinesulfone)-n'-isonicotinoylhydrazide, possess for their preparation, and pharmaceutical composition based on these salts

Info

Publication number: WO2001032650A2
Application number: PCT/RU2000/000438
Authority: WO
Inventors: Alexandr Leonidovich Reshetov; Nikolai Mikhailovich Goloschapov; Tamara Petrovna Filipskikh; Ljubov Elizarovna Kostjuk; Elena Nikolaevna Goloschapova; Elena Andreevna Michurina; Anatoly Anatolievich Zavarzin
Original assignee: Alexandr Leonidovich Reshetov; Goloschapov Nikolai Mikhailovi; Tamara Petrovna Filipskikh; Ljubov Elizarovna Kostjuk; Elena Nikolaevna Goloschapova; Elena Andreevna Michurina; Anatoly Anatolievich Zavarzin
Priority date: 1999-11-05
Filing date: 2000-11-03
Publication date: 2001-05-10
Also published as: RU2191015C2; UA72286C2; EA200200540A1; AU1423701A; WO2001032650A3

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.

Description

ALKALINE SALTS OF N-(6-ALKYL-2,4-DIOXO-l,2,3,4-TETRAHYDRO-5- PYRIMIDINESULFONE)-N'-ISONICOTINOYLHYDRAZIDE, WHICH CAN POSSESS ANTIMICROBIAL AND IMMUNOTHROPE ACTIVITY, THE METHOD FOR PRODUCING THESE SALTS, THEIR USE AS IMMUNOMODULATOR AND PHARMACEUTICAL COMPOSITION BASED ON THESE SALTS. The invention relates to the field of biologically active compounds, particularly alkaline salts of N-(6-alkyl-2.4-dioxo-1.2.3.4-tetrahydro-5-pyrimidinesulfone)-N^,-isonicotinoyl-

hydrazide, which can possess antimicrobial and immunothrope activities and can be used

as immunomodulator. as well as the method for producing these salts and pharmaceutical composition on their basis.

There is known diucifon possessing immunomodulating properties and having immu- nostimulating effect on humoral and cell immunity (author's certificate N° 459228), however this compound doesn^'t possess its own mytogenic activity but is capable of increasing T-cell proliferation only in response to phytohemagglutinin (PGA). The diucifon toxicity is

2600 mg/kg.

Also there is known N-(6-methyl-2,4-dioxo- 1,2,3 ,4-tetrahydro-5-pyrimidinesulfone)-N'- isonicotinoylhydrazide hydrate (isofon) (Russian patent JYs 2141322) possessing antimicrobial and antimycobacterial properties, but because of its low water solubility it has low biological activity that is confirmed by the experiment on animals (rabbits), when it was

shown that near 50% of isofon are detected in natural state in vital functions products.

The invention is based on the task of creating new compounds possessing antimicrobial and immunothrope activities, developing the method for producing these compounds, us- ing them as immunomodulator and creating new pharmaceutical composition on the base of these compounds.

The formulated problem is solved by offered 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.

The compounds of formula I possess antimicrobial and immunothrope activities and can be used as immunomodulator, containing the effective quantity of active substance, at chronic recurring diseases complicated with bacterial infections on the immunodeficiency background.

The formulated problem is also solved by the method for producing the compounds of formula I. This method includes mixing of isofon derivatives of general formula II

J

where R is as described above, with corresponding alkali and precipitating the obtained salt with water miscible solvent.

The formulated problem is also solved by claimed pharmaceutical composition including effective quantity of alkaline salts of formula I as an active substance and additives.

The claimed compound of formula I, where M is Na and R is methyl (isodinafon). repre¬

sents white amorphous powder, m.p. 248-250°C, highly soluble in water.

The toxicity of isodinafon was determined in Litchfield- Wilckokson sharp experiment on

white outbred mice of 18-20 g by weight and makes up over 700 mg/kg.

The claimed compound was tested in experiments on animals.

Determination of isodinafon immunothrope activity. a) The effect on humoral immunity was studied by the method of local hemolysis in gel.

Mice of FI hybrids (CBA x C₅₇BI₆) were immunized with ram erythrocytes in the dose of 5 x

10⁶ and immediately after that the tested compounds were introduced both orally in starch

suspension and parenterallv. The number of antibody forming cells (AFC) in mice spleen was estimated on the 5th day and the immune response stimulation index was determined

by ratio of AFC number in experiment group to AFC number in control group.

These data are presented in the Table 1. Table 1. The effect of tested compounds on accumulation of antibody forming cells (AFC) in mice s leen.

The immune response stimulation indexes for isofon, soluble diucifon and isodinafon while introducing these preparations inwards are indistinguishable for certain. It means that

the tested preparations have the same effect on humoral immunity that is tested in the AFC

accumulation reaction in mice spleen.

The offered isodinafon preparation works also while parenteral introducing in lower dose that allows saving this preparation. It works to the same extent as soluble diucifon. but the advantage of isodinafon is in its lower toxicity in comparison with soluble diucifon while introducing both orally and parenterally (while peroral introduction LD₅₀ for isodinafon is

7000 mg/kg and for soluble diucifon is 2600 mg/kg (2136÷3042); while parenteral intro¬

duction LD₅₀ for isodinafon is 1750 mg/kg (1286÷2380) and for diucifon is 1 100 mg/kg

(887÷1364)). b) The effect of the preparations on T-lymphocyte proliferation was studied in vitro. Human mononuclear cells were incubated for 3 hours in the presence of different concentrations of each tested preparation. The broad concentrations range from 0.01 to 5 mkg/1 was studied for every preparation. The physiological solution of 0.9% NaCl was used as a negative control.

The results of the new compounds effect on the T-lymphocite proliferation intensity in

human cells culture in vitro are presented in the Table 2.

Table 2. The isodinafon effect on T-lymphocyte proliferation in culture cells in vitro.

So the offered preparation, as well as isofon, possesses both its own mytogenic activity and the activity in response to phytohemagglutinin, but it shows its activity while adding in lower doses.

c) The effect of the preparations on phagocytic activity of macrophages was studied ac¬

cording to ink clearance in the blood from retroorbital sinus of mice which were given the preparation orally. The results were estimated according to phagocytic index (Table 3).

Table 3. The effect of preparations in study on phagocytic activity of macrophages.

The results given in the table show that isodinafon stimulates phagocytic activity of macrophages as well as soluble diucifon while any way of introduction, but it works better while oral introduction, whereas isofon works only while oral introduction. d) The lmmunocorrecting ability oi isodinafon was determined on the model of immunodeficiency induced by radiation treatment of mice FI (CBA x C₅ BI₆) in the dose of 4 Gray It was shown that radiation treatment of mice in the dose of 4 Gray decreases immune response 15-20 times, the recovery of immune response begins on the 6-8th day and finishes to the 30th day (Yarihn A A . Polushkina E F , Filatov P P. The effect of ionizing radiation on the ratio of lymphoid cells populations of mice RadiobiologN , 1976. v 16. N3, pp 451-454) While using this experimental model animals were given the

preparations in study in the dose of 500 mkg/mouse in 0.5 ml of starch suspension orally

and 100 mkg/mouse in 0.2 ml of physiological solution parenterallv The preparations were introduced three times on the 6. 7, 8th

after radiation treatment The control group of animals was given 0.5 ml of starch suspension or 0.2 ml of physiological solution On the 15th day the AFC number in animals spleen was determined by the method of local hemo- lysis in gel

The data are shown in the Table 4

Table 4. The isodinafon effect on the recovery of the process of antibody forming cells production in spleens of irradiated animals.

Using the obtained data on recover}' of immune response after radiation treatment it is possible to make the conclusion that isodinafon shows immunocorrecting activity not less than that of preparations of comparison while oral introduction, and 2 times greater than that of

isofon while parenteral introduction in the 5 times less dose.

Determination of isodinafon anti tuberculosis activity.

The activity was tested in comparison with isofon and highly effective immunomodulator,

soluble diucifon. The ordinary and tubazide resistant up to 100 mg laboratory strains of tuberculosis mycobacteria H₃₇ RV were used for infection. 230 mice of CBA line were in- fected by intravenous injection of 0.25 mg of tuberculosis mycobacteria culture in 0,5 ml of physiological solution. The experiment lasted for 2 months from September 10 till No¬

vember 12 1996. 16 groups of animals were observed, 15 mice in each, all animals were kept in the same vivarium conditions at the standard ration. During the whole experiment the control groups were given 0,5 ml of starch suspension via tube or 0,2 ml of physiological solution parenterally 5 times a week. The experiment animals were given the prepara¬

tions in different doses in 0,5 ml of starch suspension orally or in 0,2 ml of physiological

solution parenterally. As the experiment was finished all the animals, both fallen and

slaughtered, were weighted, their internals were weighted, too. and the degree of internal lesion was estimated in "+", besides the preparation effect was estimated according to average life time (ALT).

The experiment results are shown in the Table 5.

Table 5. The effect of preparations in study on anti tuberculosis activity.

*Values, reliably differing from control. PO.05

As the results show isodinafon has the pronounced anti tuberculosis effect both on myco¬

bacteria susceptible to the most of anti tuberculosis preparations and on tubazide resistant mycobacteria. Judging by the lung lesion index of infected animals treated by isodinafon

and soluble diucifon. isodinafon has greater anti tuberculosis effect than preparations in

comparison while introducing parenterally.

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

The isodinafon anti leprosy activity in comparison with isofon and soluble diucifon was studied on male mice of CBA line infected with laboratory leprosy mycobacteria strain

Cur-4. Leprosy mycobacteria were taken in the dose of 5000 and introduced intraplantary

by Shephard method. The experiment lasted for 6 months from February 7 till August 4, 1996. 5 groups of animals were observed. 20 mice in each. All animals were kept in the same vivarium conditions at the standard ration.

The first group is control: during 6 months 5 times a week the mice of this group got 0.5 ml of starch suspension via tube. In the other experimental series the control animals got 0.2 ml of physiological solution by specified dates, the experimental animals got preparations diluted in physiological solution.

After 6 months the animals were slaugtered by cervical displacement of spine, the internals were subjected to bacterioscopy analysis, no pathological deviations typical for any group

were found out. While bacterioscopy analysis the infection sites (paw) were grind in 2 ml of 0,1% albumen solution, smear was made of 0,01 ml of suspension and colored by Cill-

Nilsen. and the number of mycobacteria per mouse in each group was counted.

The results are shown in the Table 6.

As it appears from the above data isodinafon in all studied doses and application methods

has the pronounced anti leprosy effect in contrast to soluble diucifon and isofon that don't possess anti leprosy activity in the dose of 25 mkg/mouse.

Study of isodinafon antimicrobial activity.

The antimicrobial activity of isodinafon in comparison with soluble diucifon and isofon

was studied on Bacillus cereus and Staphylococcus aureus cultures. For determination of

the preparations antimicrobial effect on B. cereus 100 mg of each preparation were diluted

in 10 ml of phosphate buffer (pH 7,0) and 1 ml of the solutions was added to test-tubes containing 1 ml of molten medium No 1 (USSR State Pharmacopeia, iss. XI. ed. 1, v. 2, p.200) and 1 ml of B. cereus daily broth culture diluted a 1000 times with physiological salt solution. The contents of test-tube were immediately poured out on Petri dish contain- ing 15 ml of solidified medium No 1 , the dishes were placed into thermostat at the temperature from 30 to 35°C.

For determination of the preparations antimicrobial effect on St. aureus 100 mg of each preparation were diluted in medium No 8 (USSR State Pharmacopeia, iss. XI, ed. 1 , v. 2, p.208) and 1 ml of the solutions was added to test-tubes containing 4 ml of molten medium No 10 (USSR State Pharmacopeia, iss. XI. ed. 1. v. 2, p.208) and 1 ml of Staphylococcus aureus daily broth culture diluted a 1000 times with physiological salt solution. The con¬

tents of test-tube were immediately poured out on Petri dish containing 15 ml of solidified

medium No 10. the dishes were placed into thermostat at the temperature from 30 to 35°C. The experiment lasted for 5 days.

Failing test strains growth on appropriate nutrient mediums the preparations antimicrobial effect was recorded. The experiment results are presented in Table 7.

Table 7. The antimicrobial effect of isodinafon in comparison with soluble diucifon and isofon.

+ - microorganisms growth presence

— microorganisms growth absence

± - insignificant microorganisms growth As the experiments results show isodinafon has bactericidal effect on B. cereus microorganisms and pronounced bacteriostatical effect on St. aureus in contrast with isofon possessing no antimicrobial activity and soluble diucifon possessing low bacteriostatical activity.

Other alkaline salts of formula I show similar activity.

The method for producing the compounds of formula I is illustrated by the Example 1.

Example 1. Disodium salt of N-(6-methyl-2,4-dioxo- 1.2.3 ,4-tetrahydro-5-pyrimidinesul-

fone)-N'-isonicotinoylhydrazide.

2 g (6.2 mmol) of isofon were dissolved in 13 ml of solution containing 0,52 g (13 mmol) of sodium hydroxide. The obtained slightly yellowish solution was filtered through glass filter and the filtrate was transferred to a three-neck flask. The filtrate was cooled to room temperature and 60 ml of ethanol were added with intense stirring. The reaction was stirred

for 2 hours till the full salt precipitation. The precipitate was filtered out, washed with 20

ml of alcohol and dried in a desiccator at 50°C.

The yield was 1.7 g (74,2%) of white amorphous powder, m.p. 300°C, highly soluble in water.

The examples of pharmaceutical compositions are presented below. Injections: active substance isodinafon - 200 mg solvent - novocaine 0.5% or 0,25%) - 5 ml

Capsules: active substance isodinafon - 100 mg, 200 mg, 500 mg.

Tablets:

active substance isodinafon - 200 mg fillers: starch - 30 mg

talc - 10 mg lactose - 10 mg

Ointment:

active substance isodinafon - 2 g

base: emulsive wax - 20 g vaseline oil - 20 g

glycerin - 20 g

distilled water - 38 g.

Thus offered alkaline salts of formula I possess wide range of immunothrope activity in doses lower than that of isofon; they keep up anti leprosy and anti tuberculosis activity and

have effect on pathogenic microorganisms. Considering this the offered compounds may be used in medicine as immunomodulator in treatment of chronic recurrent diseases complicated with bacterial infections on the immunodeficiency background, as well as antimicrobial means.

Claims

Alkaline salts of N-(6-alkyl-2,4-dioxo-l,2,3,4-tetrahydro-5-pyrimidinesulfone)-N'- isonicotinoylhydrazide of general formula I:

where R is 1-4 carbon atoms alkyl and M is alkali metal.

2. Alkaline salts of N-(6-alkyl-2,4-dioxo-1.2.3,4-tetrahydro-5-pyrimidinesulfone)-N'-

isonicotinoylhydrazide of general formula I according to claim 1 , possessing antim¬

icrobial and immunothrope activities.

3. The method for producing alkaline salts of N-(6-alkyl-2.4-dioxo-l,2,3,4-tetrahydro-

5-pyrimidinesulfone)-N'-isonicotinoylhydrazide of general formula I according to claim 1 , wherein isofon derivatives of general formula II

where R is as described above, are mixed with the appropriate alkali and the obtained

salt is precipitated with water miscible solvent.

. Pharmaceutical composition, containing active substance and additives, that is characterized by containing effective quantity of alkaline salt of N-(6-alkyl-2,4-dioxo- l ,2,3,

4-tetrahydro-5-pyrimidinesulfone)-N'-isonicotinoylhydrazide of general formula I according to claim 1 as active substance.

5. Use of alkaline salts according to claim 1 as immunomodulator. containing the effec¬

tive quantity of active substance, at chronic recurring diseases complicated with bac¬

terial infections on the immunodeficiency background.