RU2000004C1 - Copolymers of 2-methyl-5-vinylpyridine and n-vinylpyrrolidone showing immunostimulation effect - Google Patents

Abstract

Usage: as immunostimulating agents in medicine. SUMMARY OF THE INVENTION: Product: copolymers of 2-methyl5-vinylpyridine and N-vinylpyrrolidone of the general formula. SHO-P where n 25-40, with an average ecos molecular molecular weight M 29 29000-45000; yield 20%; pH of a 10% aqueous solution of 6.5-7.5. Reagent 1; 2-methyl-5-vinylpyridine, Reagent 2: N-vinylallyrrolidone. Reaction conditions: heating at 65-70 ° C in the presence of azo-bis-isomeric acid dinitrile, Н202 and c-СБНе while maintaining the molar ratio of reactants 1 and 2 is -1: 32-65. 8 tab., 3 pr. 70 s

Description

The invention relates to the field of polymer chemistry, in particular to new biologically active chemical compounds - copolymers of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of the general formula 1

Jmo-n

where n is 25-40 with an average viscosity molecular weight M / 7 of 29000-45000 daltons having an immunostimulating effect

This property suggests the possibility of using compounds of the general formula 1 in medicine and agriculture.

A copolymer of 5-isopropenyl tetrachloro and N-vinyl pyrrolidone is known having the properties of an immunoadjuvant, a surface antigen of viral hepatitis B 1.

SUMMARY OF THE INVENTION New copolymers of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone have an immunostimulating effect.

Compounds of general formula 1 are prepared by the radial copolymerization of 2-methyl-5-vinylpyridine (2-M-5-VP) and N-vinylpyrrolidone-VPD) in the presence of azo bis iso as the initiator of copolymerization

o o o o o o

about

butyric acid and as a molecular weight regulator of hydrogen peroxide and cyclohexane, the process being carried out at an initial molar ratio of 2-methyl-5-vinylpyridine: N-vinylpyrrolidone equal to 1: 32-65, respectively, followed by the addition of consumable monomers in an amount necessary to maintain a molar ratio of 2 - methyl-5-vinylpyridine: N-vinylpyrrolidone equal to 1: 32-65, respectively, and the target product is isolated by precipitation in diethyl ether.

Due to the large difference in the copolymerization constants, g 2-M-5-VP / G N-VPD 000.2-methyl-5-vinylpyridine is rapidly consumed in the copolymerization process. The synthesis of a copolymer of a given composition is ensured by maintaining a constant ratio of 2-M-5-VP and N-VPD by compensating for the monomers consumed in the synthesis process with a make-up mixture containing 25-40% 2-M-5-VP and 60-75 % N- VPD. The volume of the impregnation mixture fed to the reactor was calculated every 20 minutes from an analysis of the concentration ratio of monomers in the reaction mixture. Upon reaching about 20% conversion by monomer, the reaction mixture was cooled to 20 ° C and precipitated in diethyl ether.

The structure of the obtained samples of copolymers of general formula 1 was proved by determining the composition (the number of pyridine units determined by non-aqueous titration) and C NMR spectra. To determine the molecular weight distribution of the MMP of the copolymer, size exclusion chromatography was used in combination with small-angle laser light scattering and differential capillary Viscometry

The examples below illustrate the invention,

In the examples, 2-M-5-VP with a basic substance content of 95-96% (d 0.956} and N-VPD with a basic substance content of 98.5-99.5% (d 1,040) are used.

Example 1. Preparation of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone (p 35) (Compound 1).

An initial reaction mixture containing 2.4 L (2500 g) of N-vinylpyrrolidone, 80 ml (76.5 g) of 2-methyl-5-vinylpyrilidine, 14 ml of a 30% aqueous hydrogen peroxide solution was charged into the reactor. 250 ml of cyclohexane and 10 g of azobisisobutyric acid dinitrile (ADN). The content of monomers in the reaction mixture is analyzed by chromatography. The reaction mass is heated to 65-70 ° C and while maintaining this temperature in the reactor

fed a make-up mixture containing 384 g (65 mol%) of N-VPD. 238 g (35 mol%) of 2-M-5-VP, 2.5 g of ADN and 5 silt of a 30% aqueous solution of H20a. Then the reaction mixture is cooled and precipitated in diethyl ether. After the precipitated product B is reprecipitated in diethyl ether. The target product is washed on the filter with 2 L of diethyl ether, dried at atmospheric pressure at 40-60 ° C, followed by drying at 80-90 C in a vacuum oven. 560 g of compound 1 are obtained.

Example 2. Preparation of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone (p-25) (Compound 2).

An initial reaction mixture containing 2.4 L (2,500 g) of N-vinylpyrrolidone, 44 ml (42 g) of 2-methyl-5-vinylpyridine, 15 ml of a 30% aqueous HaCte solution was charged into the reactor. 250 ml cyclohexane and 10 g azo bis isobutyric acid dinitrile (ADN). After heating the reaction mixture to 65-70 ° C for 5.5 hours, a make-up mixture containing 450 g (75 mol%) N-VPD, 172 g (25 mol%) 2-M-5-VP . 2.5 g of ADN and 5 ml of a 30% aqueous solution of H202. The reaction mixture was then cooled and further worked up as in Example 1. 565 g of compound 2 was obtained.

Example 3. Obtaining a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone (p 31) (Compound 3).

An initial reaction mixture containing 2.4 L (2500 L) of N-vinylpyrrolidone, 62 ml (59.6 g) of 2-methyl-5-vinylpyridine, 14 ml of a 30% aqueous solution of Y202, 250 ml of cyclohexane was charged into the reactor. 10 g of aero-bis-isobutyric acid (ADN). After heating the reaction mixture to 65-70 ° C for 5.5 hours, an impregnation mixture containing 410 g (69 mol%) of N-VPD is fed into it. 212 g (31 mol%) of 2-M-5-VP, 2.5 g of ADN and 5 ml of a 30% aqueous solution of H2U2. The reaction mixture was then cooled and further worked up as in Example 1. 562 g of compound 3 was obtained.

Example 4. Obtaining a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone (p 40) (Compound 4).

An initial reaction mixture containing 2.4 (2500 g) N-vinylpyrrolidone, 100 ml (95.8 g) 2-methyl-5-vinylpyridine, 14 ml of a 30% solution of H202, 250 ml of cyclohexane and 10 g is loaded into the reactor. dinitrile azo-bis-isobutyric acid (ADN). After heating the reaction mixture to 65-70 ° C for 5.5 hours, a make-up mixture containing 352 g (60 mol%) of N-VPD, 270 g (40 mol%) of 2-M-5-VP is fed into it. .2.5 t ADN and 5 ml of a 30% aqueous solution

2.5 g of LDN and 5 ml of a 30% aqueous solution of H202. then the reaction mixture was cooled and further worked up as in Example 1. 558 g of compound 4 was obtained.

All products obtained according to Examples 1-4 are white amorphous powder. They are soluble in water, the pH of a 10% aqueous solution of compounds 1-4 is 6.5-7.5.

Characteristics of the compounds obtained are presented in table 1.

Biological tests.

Determination of acute toxicity of compounds 1-4.

The test was performed on healthy mice with FI (CBAx Cs7Bi) hybrids weighing 18-20 g. The test compounds were dissolved in a sterile solution. Test dose of 0.4 ml of a 10% solution in isotonic NaCI. Observation time 7 days. The drug is considered to have passed the test if none of the 5 experimental mice die during the intended observation. It was not possible to establish LDso for mice, since even at a dose of 5000 mg / kg, no death of mice was observed. Thus, compounds 1-4 can be classified as non-toxic substances. In addition, it was found that compounds 1-4 at a dose of 500 mg / kg did not cause irritation and hair loss in white mice by subcutaneous administration.

The study of the immunostimulating effect of compounds

1-4 in vitro.

The immunostimulating effect of In vitro was studied by the method of Migration activity of a colony of formed spleen cells-endogenous colony formation. For this, mice were irradiated in cassettes of 30 mice with y-rays of cesium -137 with an exposure dose of 600 X-rays at a dose rate of 22.79. Solutions at a dose of 50 mg per mouse were injected subcutaneously into the surrounding withers 24 hours before irradiation. Mice were killed on day 10. The spleens were removed and placed in a Boen fixative. Colonies were counted visually under a magnifying glass. The immunostimulating effect of the polymers was taken into account by the difference in the number of colonies compared with the control (physiological saline at a dose of 0.5 ml). The immunostimulant prodigiosan known in medical practice (2) was used as a reference standard. The test results are presented in Table 2.

It can be seen from Table 2 that all tested compounds 1-4, like the well-known prodigiosan preparation, have a pronounced immunomodulatory effect. The most active is compound 1, which, like prodigiosan, causes the formation of more than 35 colonies, i.e. confluent colony growth, when almost all colony formation is observed on almost the entire surface of the spleen.

In vitro study of the immunostimulating effect.

Compounds 1 and 2 were studied for immunostimulatory effects.

0 in animal experiments. The following pathogens of infectious diseases of agricultural animals were used in the experiments: virulent virus of classical plague., Pigs (CSF) in the form of culture

5 fluids with an infectious titer of 6.5-7.0 KKID5o (culture-cell infectious doses) and the causative agent of anthrax in spore and vegetative forms.

The immunostimulating effect of compounds was studied on telts and sheep during Siberian ringing and gilts during classical swine fever.

Bodies there (black-and-white breed, weight 140-150 kg) were administered subcutaneously about

5 dose of 0.34 mg / kg in a volume of 1 ml containing 100 thousand spores, sheep (Prekos breed, weight 25-30 kg) in the same dose in a volume of 0.5 ml containing 40 thousand spores of anti-vaccine vaccine from strain 55. For guinea pigs (Bela breed is large, weight 25-30 kg), the compounds were administered intramuscularly at a dose of 1 mg / kg in a volume of 2 ml in a mixture with an inactivated vaccine against classical swine fever. Control eradication was carried out on day 1214, a calf at a dose of 200 million spores was orally administered as a bolus, and sheep subcutaneously at a dose of 1 million spores in a volume of 0.5 ml of virulent strain 76 of the causative agent of anthrax. Gilts infected

0 intramuscularly (intramuscularly) with a virulent strain of the virus of classical swine fever at a dose of 100 thousand culture of "o-cell infectious doses in a volume of 1 ml. The control groups of animals, which

5, the same doses of the vaccine were administered in saline, and the virulence control groups of the materials used for infection.

The results of studying the immunostimulating effects of the compounds in combination with the anti-ulcer vaccine from strain 55 in an experiment on calves are presented in Table 3.

From the data of table 3 it is seen that compounds 1 and 2 at a dose of 0.34 mg / kg of mass at

In a single administration with the vaccine, on the 14th day after vaccination, intense immunity is established in t calves, while administration of the vaccine in saline at the same time creates immunity in only 75.0% of the animals. Standard vaccination dose

vaccines from strain 55 to create a strong immunity should contain 2.5 x 107 spores, whereas when using compounds 1 and 2, 100% immunity is created with a dose of 1 x 105 spores. those. 250 times smaller.

The results of the study of the immunostimulating effect of compounds 1 and 2 in combination with the antisythera vaccine in a sheep experiment are presented in Table 4.

From the data in Table 4 it is seen that using compounds 1 and 2 to create 100% intense immunity, a dose of 4 x 104 spores is eliminated, while according to the instructions for use of the vaccine from strain 55, one vaccination dose should contain 1x10 spores. those. 250 times higher. Sheeps of group 3, which were injected with the vaccine in saline solution, were all ill with high fever, there was a rejection of food, water and severe oppression. 7 out of 12 goals.

The results of a study of the immunostimulating effect of Compound 1 on gilts in classical swine fever (CSF) are presented in Table 5.

From the data in table 5 it can be seen that the combined use of compound 1 and an inactivated vaccine against classical swine fever at a dose of 2 ml creates a strong immunity in all vaccinated animals on the 14th day after the vaccine, while the double administration of this vaccine with an interval of 7 days in a dose of 2 ml (total volume 4 ml) did not create intense immunity, animals were ill with a temperature reaction, refusal to feed was observed, which significantly affects gain.

Consequently, the administration of Compound 1 allows for the creation of intense immunity when administered once with an inactivated vaccine in a dose of 2 ml already on the 14th day after vaccination. Compound 1 has an immunostimulating effect when combined with both a live vaccine (with Siberian fever) and an inactivated vaccine (with classical swine fever).

In addition, the therapeutic and prolonging effect of compounds 1, 2, and 3 in combination with chemotherapeutic agents and antibiotics (with Siberian ringing) and inactivated vaccines (with classical swine fever) was investigated.

In addition, the therapeutic and prolonged effect of compounds 1, 2, and 3 in combination with chemotherapeutic agents and antibiotics in the Siberian ring in white mice and compound 1 in sheep and rabbits was investigated. The sheep were infected with virulent strain 76 subcutaneously at a dose of 1 million spores in a volume of 0.2 ml. rabbits (weight 2.5-3.0 kg) - in the same dose in a volume of 1

ml, white mice at a dose of 100 spores in a volume of 0.2 ml. The complex of drugs was administered 24 hours after infection at a time in a daily dose for 7 days. The complex of compound 1 with chemotherapeutic agents was administered with food 1 day before infection and 10 days after infection at a time in a single daily dose.

As a reference standard, the known prolongator, ecmoline, was used.

5 3.

The control was groups of animals that were injected with antibiotics in physiological saline.

The effectiveness of the treatment and prophylactic and prolonged action of the compounds in combination with antibiotics and chemotherapeutic drugs was evaluated by the percentage of survival and disease compared with the control. The specificity of the death of animals was confirmed by pathological examination and identification of pathogens. The presence of post-infection immunity was determined by the level of neutralizing antibodies and the response to control of my infection.

The results of the study of the therapeutic, prophylactic and prolonging effect of compounds 1 and 2 in combination with antibiotics in Siberian rats on white mice with a single administration of a daily dose for 7 days are presented in Table 6.

From the data in Table 6, it can be seen that compounds 1-3, when administered in combination with antibiotics for 7 days, protect against the death of 80% of mice infected with the causative agent of anthrax with 100% death in the control.

The results of studying the therapeutic effect of antibiotics in combination with compound 1 in comparison with antibiotics dissolved in physiological saline on sheep infected with anthrax are shown in Table 7.

0 From the data of table 7 it is seen that when

in the treatment of sheep with a complex of compound 1 with antibiotics tetracycline and streptomycin, the clinical signs of the disease were completely absent, while

On the administration of antibiotics in physiological saline, all 3 heads of sheep of the 2nd group showed a pronounced temperature reaction, inhibition, refusal of feed, which significantly affects the yield of livestock production.

The results of treating bacterial infections of fur animals with the complex of compound 1 and antibiotics of the fluoroquinolone series, namely, 1 - ethyl-6-fluoro-7- (4-methyl-1-piperazinyl) -1,4-dihydro-4-oxa -3- quinoline carboxylic acid (compound 4) 4 and its phosphonoacetic acid salt (compound 5). For the treatment of anthrax in rabbits, see table 8.

From the data of Table 8 it can be seen that in groups 1 and 2, where compound 1 was used as a prolongator, and in groups 3 and 4, where ecmoline was used. 17 rabbits out of 20 survived at 100% death in control, which indicates the possibility of using compound 1 as a prolongator in combination with new generation antibiotics based on fluoroquinolones to treat infectious diseases

bacterial nature of fur-bearing animals on the example of the treatment of anthrax in rabbits.

All the data presented on the study of the immunostimulating effect of the compounds show:

β-compounds at a dose of 0.34 mg / kg in combination with the vaccine from strain 55 have a pronounced immunostimulating effect in Siberian stink in animals (calves, sheep), which makes it possible to reduce the vaccine dose to create intense immunity in Siberian stink by 250 times ;

-the combined use of compound 1 at a dose of 1 mg / kg and an inactivated vaccine against classical swine fever creates intense immunity 14 days after vaccination and allows a 2-fold reduction in the dose and frequency of administration of the vaccine;

Compound 1 at a dose of 4 mg / kg weight (sheep) and 10 mg / kg weight (rabbits) in combination with antibiotics have a pronounced therapeutic effect in Siberian ringing. It can be used as a prolongator.

Thus, copolymers of 2-methyl-5-vinylpyridine and stimulants for use in medicine and agriculture.

Claims (1)

SUMMARY OF THE INVENTION Copolymers of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of the general formula cng-sn Characteristics of the resulting compounds where n 25-40, with an average viscosity mol.m M 29000-45000 D, possessing an immunostimulating effect. Table 1 Medium viscosity molecular weight; M w is the weight average molecular weight; M p is the number average molecular weight. Table 2 The results of the study of the immunostimulating effect of compounds 1-4 In vlrto Immunostimulating effect of compounds 1 and 2 in case of anthrax Continuation of table 1 Continuation of Table 2 Table 3 Immunostimulating effect of compounds 1 and 2 in Siberian ringing in sheep Continuation of Table 3 Table 4 Continuation of table 4 15200000416 Table 5 Immunostimulating effect of compound 1 on gilts in CSF Note: INVAK - inactivated vaccine,  - temperature reaction, oppression, refusal of feed The therapeutic and prolonging effect of compounds 1-3 in combination with antibiotics for Siberian zve on mice Continuation of Table 5 Table 6 Table 7 Therapeutic and prolonging effect of compound 1 in combination with antibiotics for Siberian ringing in sheep Continuation of Table 6 Continuation of Table 7 Therapeutic effect of compound 1 and fluoroquinolones in Siberian rabbit ringing Table Continuation of Table 8