Salt of bis(oxymethyl)phosphinic acid with isonicotinic acid hysrazide (tubophen) having antituberculosis action and method for production thereof

FIELD: organic chemistry, medicine.

SUBSTANCE: disclosed are bis(oxymethyl)phosphinic acid salt of formula I and method for production thereof.

EFFECT: antituberculosis agent of decreased toxicity without losses of therapeutic activity.

3 cl, 3 dwg, 9 tbl, 1 ex

 

The invention relates to the chemistry of nitrogen-containing heterocyclic compounds and organophosphorus compounds, namely salts of bis(oxymethyl)-phosphinic acid with isonicotinic acid hydrazide of the formula I (hereinafter referred to as tobien"), which can be used as a drug in the veterinary and medical practice for the prevention and treatment of tuberculosis. The claimed compound, its properties and the method of obtaining not described in literature.

There are a large number of anti-TB drugs, and their modern classification is based on the effectiveness of their influence on the agent. In the classification are the most effective drugs: chemotherapy drug isoniazid (GINK) and the antibiotic rifampin, drugs average efficiency: antibiotic streptomycin, kanamycin, florimitsin (viomycin), cycloserine, drugs, ethambutol, ethionamide, protionamide, pyrazinamide (isamid) and drugs of moderate efficiency: PASK (n-aminosalicylic acid), Tiron (Thiacetazone).

Despite the large number of anti-tuberculosis drugs, isoniazid, representing isonicotinic acid hydrazide, is the best of the existing drugs and is included in almost all schemes for the prevention and treatment of tuberculosis of animals & people (Wiesel A.A. Treatment of tuberculosis of the respiratory organs. Kazan, HPVEE "Salamat", 1998, 119 S.), (Mashkovsky PPM Medicines. Vol.2. Kharkov, 1998, s-333), (Smolyaninov SCI, NIKOLAY Koshcheev. Specific prevention of tuberculosis in young cattle.// Sat. scient. Tr./ VNIIBT. - Omsk, 2001: Infectious pathology of animals. C. 168-170).

Known structural analogues of the claimed compounds. The orotate the hydrazide of isonicotinic acid shows antimycobacterial and immunotropic activity (U.S. Pat. EN 2044728, publ. 27.09.1995). Melamine salt of bis(oxymethyl)phosphinic acid is a regulator of growth and development of plants (U.S. Pat. EN 2158735, publ. 10.11.2000). However, isoniazid is the closest structural analogue of the present invention, having the same type of activity.

It is known that isoniazid used for the prevention and treatment of tuberculosis, is not free from side effects and as a pharmacological tool has a certain biological activity, can cause changes in the indicators of metabolic and structural-functional state of organs and systems, and in overdose accompanied by poisoning and fatal (Saga L.A. Biochemical rationale for ways to reduce the hepatotoxicity of isoniazid on the basis of the combination with polysaccharides: author. descend. Pharm. Sciences/ Pyatigorsk Gosford. and ADEME. Pyatigorsk, 1999, 21 C.). It is important to note that the toxicity of isoniazid is associated mainly with its metabolites (I.E. Roller, Smania CENTURIES Essence of hepatotoxic action of isoniazid.// Nursing, 1984, No. 1, 49-52), so the synthesis of an analogue of isoniazid, perhaps with a different type of metabolism, leading to reduction of toxicity, is of particular interest.

The purpose of the invention is a new, more effective compounds with anti-TB activity, do not have hepatotoxic actions contributing to the rapid recovery, expanding Arsenal of known drugs, and how to obtain it.

The technical result of the invention is to reduce the toxicity of the drug while maintaining a high therapeutic efficacy and the absence of local and General reactogenicity means at therapeutic doses.

The technical objective is achieved by the invention, namely salt bis(oxymethyl)phosphinic acid with isonicotinic acid hydrazide of the formula I (turbofan) and method of reception. The essence of the proposed method of obtaining tubiana lies in the interaction of bis(oxymethyl)phosphinic acid and the hydrazide of isonicotinic acid in methanol under stirring, followed by heating to 60°With, by keeping to matnog temperature for 12-15 h and separation of precipitated sludge conventional techniques. For a better understanding of the invention examples of obtaining the claimed salt and the results of the study it antitubercular properties.

An example of a specific implementation.

To the suspension to 7.61 g (by 0.055 mole) of the hydrazide of isonicotinic acid in 40 ml of methanol at room temperature was added dropwise a solution of 7.75 g (0.061 mol) of bis(oxymethyl)phosphinic acid in 40 ml of methanol. The reaction mass is stirred, reaching full transparency of the solution, then heated to 60°and leave for 12-15 hours Sucked fallen during this time, the product and dried. Obtain 8.8 g (60,2%) of the product salts bis(oxymethyl)phosphinic acid with isonicotinic acid hydrazide as a white crystalline substance. TPL 109-110°C. NMR31P (36,48 MHz, D2O): δPAt 37.20 ppm Found, %: C 36.40; N, 5.28; N, 15.64; P 10.88. C8H14N3PO5. Calculated, %: 36.50; N, 5.32; N at 15.97; R 11.79.

A comparative study of the effectiveness of tubiana - salt bis(oxymethyl)phosphinic acid with isonicotinic acid hydrazide and isoniazid for tuberculosis was conducted for two years at 180 animals (24 white mice, 76 outbred rats and 80 Guinea pigs).

The bacteriostatic activity of tubiana and isoniazid has been studied in experiments in vitro on 2 kinds of Mycobacterium tuberculosis, drug concentrations from 10 to 0,018 mg/ml using the method is using serial serial dilutions (Pershin GN. Methods of experimental chemotherapy. M, "Medicine", 1971, s-192).

For titer activity of the studied substances took it the largest breeding, or the lowest concentration that completely inhibited the growth of Mycobacterium tuberculosis. The main parameters for evaluating the effect of tubiana and isoniazid was the ability of organisms to grow and reproduce. Evaluation of the growth properties of mycobacteria was determined by the speed and the number of grown colonies. The estimate of the number of colonies was determined in crosses scheme proposed Hendersoni: abundant growth ( +++ ), in-depth growth of strains less abundant sediment smaller lumps culture less than in the control (++); deep growth in the form of invisible sediment grain fine in a small amount of (+); full bactericidal effect, seeded culture gives no growth (-). The results of the study bacteriostatic activity of anti-TB drugs is presented in table 1.

From table 1 it is seen that the bacteriostatic activity of tubiana high and is not inferior to that of isoniazid. Turbofan at a concentration of 0.075 mg/ml had a full bacteriostatic overwhelming effect on the strain 14 M. bovis and strain M. tuberculosis H37Rv as pure nutrient medium, and adding a 10% solution of horse serum. Concentration tubiana 0,037 and 0,018 mg/ml with the same W what MMA, on the environment Sotona and environment Sotona containing 10% solution of horse serum, had only a partial inhibition of the multiplication of tubercle bacilli in the form of slightly noticeable sediment with small grains.

In turn isoniazid had a full bacteriostatic effect on the strain 14 M. bovis and strain H37Rv .tuberculosis only at concentrations of 0.15 µg/ml And strain H37Rv .tuberculosis proved to be more resistant to isoniazid, than strain 14 .bovis, and the concentration 0,018 mg/ml, the growth of M. tuberculosis did not differ from their growth in test tubes as on the environment Sotona, and adding a 10% solution of horse serum. Concentrations of isoniazid 0,075; 0,037 and 0,018 mg/ml on the environment Sotona and environment Sotona containing 10% solution of horse serum, had only a partial suppression of the reproduction of M. tuberculosis strain 14.

In the control crop growth of the test cultures was abundant, characteristic for each species of mycobacteria.

Thus, according to modern classification (Henderson, 1971) new anti-tuberculosis drug - turbofan should be classified as highly active anti-TB drugs on bacteriostatic action.

To study the bactericidal action of tubiana also used the method of successive serial dilutions. A suspension of Mycobacterium tuberculosis strain is 14, standardized by the standard BCG, were made in tubes with nutrient medium Sotona containing different concentrations of tubiana. The tubes were placed in a thermostat at a temperature of 37°and through 1,3,6,24 hours and 2.5 and 8 days bacterial content of the tubes was transferred into centrifuge tubes and centrifuged at 3000 rpm for 1/4 hour. The liquid was decanted, and the residue culture laundered from drug saline solution followed by centrifugation. The residue after removal of the saline solution was perseval 2 tubes with Lowenstein-Jensen medium. Sowing was kept in a thermostat at a temperature of 37°C for 2.5 months. The absence in this period separate or confluent colonies of Mycobacterium tuberculosis testified to the bactericidal action of the respective concentrations of tubiana. The results of the study of bactericidal activity of tubiana reflected in table 2.

From table 2 it is seen that tobien in a minimum concentration of 0.75 μg/ml provides a complete bactericidal effect on strain 14 Mycobacterium tuberculosis bovine species, only when the maximum contact time is 8 days. When exposed to a strain of Mycobacterium minimum concentration of the drug for 5 days on nutrient Lowenstein-Jensen medium were found only a few colonies (partial is bactericidal effect).

Partial bactericidal effect of tubiana first started to appear after 6 hours of contact of the drug with a strain of M. tuberculosis at concentrations of 50, 25 and 12.5 μg/ml total loss of all Mycobacterium tuberculosis bovine species occurred after 5 days at concentrations of 6.2 µg/ml With a maximum term exposure of tubiana with Mycobacterium tuberculosis (8 days) noted marked bactericidal effect of the drug in all experimental concentrations. In all the test tubes were marked by a continuous growth of M. tuberculosis bullish view.

Determination of the parameters of acute toxicity of tubiana on white mice and rats of both sexes was performed using the methodology You (Stupnikov A.A. Toxicity of herbicides and arboricides and prevention of poisoning in animals. L., "Kolos", 1975, s-219). The results of studies of acute toxicity of tubiana on white mice are shown in table 3. Parameters of acute toxicity of an aqueous solution of tubiana after intragastric administration white mice were: maximum tolerated dose (MTD) of 400 mg/kg; sredneseriynoe dose (LD50) - 668±63 mg/kg; confidence interval General (DIG) average LD50- 668 (538-798) mg/kg; LD100- 1000 mg/kg In accordance with Hygienic classification of pesticides on the basic parameters of the hazard is (Liedel, Usigan, Eyio, 1986) turbofan is a substance having a medium toxicity. By hazard class III, hazardous chemicals (GOST 12.1.007.76). In turn Spolin (Bulavin S. p. Pharmacological characteristics of tubazide.// Bull. VIEW. M., 1982. VIP, pp.61-62) found that isoniazid is highly toxic compound for mice - LD50equal 178,0±6,79 mg/kg of body weight.

The results of studies of acute toxicity of tubiana on white rats is presented in table 4. The toxicity parameters of an aqueous solution of tubiana for white rats was: the maximum tolerated dose of 4500 mg/kg; sredneseriynoe dose (LD50) - 5675±245 mg/kg; confidence interval General average LD50- 5675 (5195-6155) mg/kg; LD100- 7000 mg/kg In accordance with Hygienic classification of pesticides on the basic parameters of harm" (Liedel, Usigan, Eyio, 1986) offer a means of TB is low toxic compound. By hazard class IV, slightly hazardous chemicals (GOST 12.1.007.76).

To study the cumulative properties of tubiana used sub-chronic test R.Lim and others (Lim R., Rink K., Glass H., et al. A metod for the evalution of cumulation and tolerance by the determination of acute and subchronic median effective doses.// Arch. intern. Pfrmocodyn. 1961. V.130. P.335-336). The results are presented in table 5. The coefficient is umalali offer TB funds amounted to 4.72 in. According to currently accepted classification (Liedel, Usigan, Eyio, 1986) turbofan corresponds to substances with a moderate accumulation.

Allergenic properties of the drug was studied in accordance with the "guidelines for the formulation of research on hygienic normalization of industrial allergens in the air of working zone, approved by the Ministry of health of the Russian Federation in 1997 (Requirements for the production of experimental studies on justification MPC industrial chemical allergens in the air of working zone and atmosphere. HOWTO 1.1.578-96. The Ministry Of Health Of Russia. - M., 1997. - 16 S.). Detection of sensitization was performed using the reaction of specific lysis of blood leukocytes (RSL). Figure RSL animals first experimental group, sensitized by tubiana dose of 50 μg per animal, was 1.19%, while the second experimental group, sensitized at a dose of 200 μg per animal of 2.23%. So RSL regarded as negative, as the rate of lysis of leukocytes in both groups of experimental animals was less than 9%.

The analysis of the researches showed that the proposed anti-tuberculosis drug with intradermal sensitization of Guinea pigs has no allergenic properties.

Embryotoxic and teratogenic effects, means were assessed in matched with the accordance with the "guidelines for the study of the embryotoxic action of pharmacological substances and their influence on reproductive function" (guidelines for the study of the embryotoxic action of pharmacological properties and their influence on reproductive function. Approved by the pharmacological Committee of Ministry of health USSR 14.03.86. - 18 C.) for 20 purebred female white rats Mature age. Animals were divided into 2 groups of 10 rats each. The first experimental group received turbofan intragastrically at a dose component from 1/20 LD50- 283,75 mg/kg of body weight for 20 days. Second, the control group in similar doses received solvent (distilled water). On the 20th day of pregnancy through decapitate were killed 5 rats from each group for the study of embryonic material. Evaluation criteria embryotoxic action of the drug were used as indicators of the number of yellow bodies in the ovaries, implantation, live and dead fetuses was determined craniocaudal fruit size and diameter of planet. The formula was determined by the following indicators:

total embryonic mortality - b/×100;

preimplantation death of zygotes - B - (A+B)/×100;

postimplantation embryos death - B/A+B×100;

where a is the number of live embryos; B - the number of dead embryos; the number of yellow bodies of pregnancy.

To identify teratogenic effect of experimental and control animals were killed at the end of pregnancy. The fruits were weighed, measured their length. Research was performed under a binocular magnifying glass, placing the fruits in h is the loud Petri with saline solution. Then the fruits were divided into two groups, one was fixed in a fluid Buena during the week and used for layered evaluation of macro-structure of internal organs according to the method of Wilson (Willson, 1965) in the modification of the Department of embryology, Institute of experimental Mineralogy of the Academy of medical Sciences of the USSR. The second was used to study skeletal development method Dawson (Dawson, 1926), modified in the Department of embryology, Institute of experimental Mineralogy of the Academy of medical Sciences of the USSR (Melanin L.P. Veterinary medicines. M: Agropromizdat, 1988, s-258). Indicators embryotoxic activity tubiana presented in table 6.

The analysis of the researches showed that tobien no effect on the reproductive qualities of rats. Indicators pre - and postimplantation loss in the experimental and control animals were similar. The number of yellow bodies and places the implant in the groups was roughly equal and did not differ by more than 5.8%. Dissection of female albino rats at 20 days of gestation showed almost equal to the number of live and dead fetuses in both groups.

The study of teratogenic effects of tubiana physical examination extracted from the uterus of fruits animal experimental and control groups, visible morphological changes were observed. In the study, the status of the internal organs of the fruit by the method of Wilson, congenital malformations, and the violation of their topography is not installed.

As cooking is the courthouse square fruit according to the method of Dawson conducted a visual examination under a binocular magnifying glass. In the result, it was found that the topography of the bone and cartilage bookmarks in the skeleton of the fruit of the experimental group is not disturbed. Violations of ossification of the bones of the skull, shoulder, pelvic girdle limbs and deviations in the structure of the skeleton is also not installed.

The study of disorders of embryonic development, manifested in the postnatal period, were performed on 5 rats, from which was obtained the offspring. Analysis of the results showed that tobien no effect on body weight and duration of pregnancy in females of the experimental group (table 7).

Also did not significantly change the performance of postnatal mortality and weight gain of rats during the first 4 weeks of their life (table 8).

Thus, the main indication for embryotoxic and teratogenic effects of tubiana the experimental and control animals were in close limits and showed the absence of embryotoxic and teratogenic effects of tubiana.

The study of the tuberculostatic activity of tubiana conducted in an experimental model of tuberculosis of Guinea pigs, which were divided into 8 groups, 6 groups of experimental and 2 control. Animals 6 experimental and one control groups were infected 20 day titrated by culture of Mycobacterium tuberculosis bovine species (strain 14). The bacterial mass is based 015 mg per animal were injected subcutaneous tissue of the right inguinal region in 0.5 ml of physiological solution.

Chemoprophylaxis of tuberculosis of Guinea pigs began to be made the next day after infection. Animals six control groups orally using atraumatic tip-olive, worn on the syringe, daily entered the solutions proposed-drug - tubiana and isoniazid. And the Guinea pig first experimental group received turbofan at a dose of 20 mg/kg, the second - 10 mg/kg, and the third is 5 mg/kg, IV - 2.5 mg/kg, the fifth of 1 mg/kg and the animals of the sixth group received isoniazid preventive dose of 10 mg/kg of body weight. Animals 7 and 8 control groups received daily oral solvent (distilled water).

The experience lasted 60 days. The animals were clinical observations, conducted hematological studies, as well as allergic TB testing.

The results of prophylactic properties tubiana and isoniazid in experimental tuberculosis of Guinea pigs is presented in table 9.

The data obtained show that during the experiment all animals 1, 2, 3, 4, and 6 experimental groups survived, while 3 Guinea pigs from the 5th experienced receiving turbofan dose of 1 mg/kg and all animals 7-th control group (infected, untreated) were killed.

The result of an allergic research Guinea pigs set the go, what animals 1, 2, 3, and 6 experimental groups did not respond to the injection of tuberculin either 30 days or 60 days after their infection with Mycobacterium tuberculosis bullish view.

In Guinea pigs 4 and 5 groups receiving turbofan at doses of 2.5 and 1 mg/kg, and the animals 7-th control group after 30 and 60 days after infection was observed pronounced allergic reaction in the places of the introduction of PPD tuberculin for mammals (standard solution), which was manifested in the form of hyperemia of the skin where the injection of the allergen and education compacted swelling with a diameter of more than 5 mm

A negative result of allergic studies also were observed in 8 of the control group.

When controlling the slaughter of two Guinea pigs from 1, 2, 3, and 6 control groups after 30 days of infection in internal organs and tissues pathological changes characteristic of tuberculosis, not detected. In a laboratory study of pathological material from animal data groups to highlight original culture of Mycobacterium tuberculosis failed.

In Guinea pigs the 4th experimental group there were single tuberculous foci in the liver and lungs and enlargement of the regional lymph nodes to the size of a large pea in the areas of infection in animals with Mycobacterium tuberculosis. Animals of the 5th experimental and 7 control GRU the item after 30 days of infection noted a sharp increase in liver and spleen, contains multiple TB foci. In the lungs was also observed single occupied rash. Regional lymph nodes were enlarged and reached sizes of beans. In a laboratory study of pathological material from animals 4 and 5 experimental groups, as well as from the 7th of the control group received a positive result from all animals was highlighted in the original culture of Mycobacterium tuberculosis.

All animals from the control and experimental groups who received ARV prophylaxis (60 days)were subjected to slaughter and conducted the postmortem, bacterioscopic, bacteriological and histological examination.

On the basis of pathological and bacteriological studies have established the average total index organs and Visavuori Mycobacterium tuberculosis. The first three experimental groups receiving turbofan in doses of 5, 10 and 20 mg/kg, and 8 intact group this index was equal to 0/0. In the study of pathological material from the 4th group received turbofan at a dose of 2.5 mg/kg, the average total index organs and Visavuori mycobacteria was 6,45/6,65, 5-th control group receiving turbofan dose of 1 mg/kg of body weight, this figure was higher 17,06/12,46 and almost reached that of the 7-th control group (infected, untreated) 20,2/12,8.

<> In the study of pathological material from the 6-th control group who received isoniazid at a dose of 10 mg/kg, in 2 of 8 Guinea pigs showed a significant increase in regional right inguinal lymph nodes. However, when sown on the last nutrient Lowenstein-Jensen medium for the growth of mycobacteria was not observed, and the average total index organs and inoculation of bacilli in Guinea pigs of this group was 0.37/0.

Thus, studies have shown that the proposed anti-tuberculosis drug showed rather high preventive effect and has expressed tuberculostatic properties at doses of 5 and 10 mg/kg weight of the animal. However, isoniazid at a dose of 10 mg/kg was less active and in 2 out of 8 cases of experimental tuberculosis in Guinea pigs do not adequately demonstrated a preventive effect.

For a comparative evaluation of the impact of isoniazid and tubiana on the structure of the liver was used 20 goals Guinea pigs, divided into 3 groups, infected subcutaneously M. bovis (strain 14) at a dose of 0.15 mg bacterial mass per 1 kg of weight of the animal. Guinea pigs of the first group are not subjected to the action of therapeutic drugs. Infected animals of the second group on the second day after infection under what was Argalis daily oral introduction of isoniazid preventive dose 10 mg/kg for 2 months, and cavies third group were injected daily oral turbofan at a dose of 10 mg/kg during the same period. Slaughter of animals tested was performed at the expiration of 60 days after the start of application of the tested tools. For histological studies of the liver slices were fixed according to the method Lilly, ethanol-formalin (9:1), neutral 10%formalin. Histological sections of a thickness of 7 μm were stained with hematoxylin-esinem on Mallory, azure II-eosin, acid mucopolysaccharides were identified toluidine blue (Merkulov, G. A. Course patrologicheskoe technology. L.: "Medicine", 1969, 423 S.).

The results of morphological studies have shown that in animals of the first group of pathological changes in the liver manifested by mixed type in the form of co-location in the body granulomatous milliary nodules and lesions with predominance of necrotic changes that characterized the wave during the infection process in the body. Infectious immunopathological reactions especially clearly manifested in the microvasculature bartalini organ system. They stood out as Muhiddin, fibrinoid the swelling of the walls of blood vessels, and complete disorganization of the entire thickness of their walls. In perivascular areas unevenly located mononuclear the e cells, on the periphery of which is noted fibroblasts with signs of a sharp decline in synthetic activity. Long, active during the infection process in the body combined with hepatocellular pathology in the form of severe destructive processes, culminating galinova droplet degeneration, fatty decomposita, necrobiosis and necrosis (figure 1).

Got infected with Mycobacterium tuberculosis and subjected to prolonged exposure to isoniazid Guinea pigs has been a sharp decrease in severity of destructive phenomena characteristic of infectious process in its active phase currents. The drug inhibited the severity of allergic reactions of immediate hypersensitivity and to a lesser extent the delayed hypersensitivity type. In this regard, the structure of the liver was noted a sharp decrease in the number and area of necrosis and the surrounding area specific granulation stood out sharp vacuum cells. Lymphoproliferative, fibroblastic reaction in the structure of the mesenchymal tissue of the body had moderate intensity that characterized the long period of formation processes of separating foci of necrosis from the surrounding tissue of the body. However, the effect of isoniazid had expressed hepatotoxic effect, exemplified by the formation of the of new foci of destructive processes in reversible and irreversible degenerative changes, becoming foci of necrosis (figure 2), which significantly reduced its prophylactic efficacy.

Most prophylactic efficacy had claimed means - turbofan. After 60 days of their use in infected Guinea pigs in the liver were observed complete inhibition of the manifestations of immediate hypersensitivity and delayed type. The tuberculostatic action of tubiana prevented formation in liver foci of necrosis and the formation of specific granulomas, testified to minimize degradation processes at the earliest stages of disorganization of the components of the connective tissue and elements of the parenchyma of the organ. Irreversible destruction of mycobacteria stimulated fibroblast processes, mainly in the course of the vessels bartalini organ system, which previously was located lesions. They noted the intensive expansion of argyrophilic and collagen fibers, and in the walls of adjacent blood vessels - the lack of reaction infectious allergies. In the liver of these animals noted the lack of proliferation of connective tissue in the sinusoids, areas of edema around them, the solid line the basement membrane of capillaries, which in combination with small stellate reticuloendothelial reflect a high level of transcapillary exchange in the body. Away the signs of severe toxicity tubiana and its biotransformation products in comparison with isoniazid contributed to low manifestations hepatocelular pathology (figure 3). Upon completion of the course of chemoprophylaxis by tubiana lacked not only the secondary foci of necrosis that occurs after the use of isoniazid, but also degenerative processes irreversible.

Identified structural changes indicate low levels of damaging effects on the liver parenchyma of tubiana (compared with isoniazid), and its high performance tuberculostatic action can effectively prevent this infection.

Table 1.

Indicators bacteriostatic activity tubiana and isoniazid in the experiment in vitro.
Name of productThe test-cultureThe presence of serumThe minimum concentration of drug, mg/mlControl
1052,51,250,60,30,150,0750,0370,018
TurbofanM. bovis strain 14---------1++++++
Turbofan +--------1++++++
Isoniazid--------1+++++++
Isoniazid+-------1+++++++
TurbofanM. tuberculosis strain H37Rv---------1++++++
Turbofan+--------1++++++
Isoniazid--------1++++++++
From NIAID +-------1++++++++
Note:1- titer activity.

Table 2.

Indicators of bactericidal activity of tubiana in the experience of in vitro
The contact time of the culture and preparationThe drug dilution, mg/mlControl
502512,56,23,11,50,75
1 hour++++++++++++++++++++++++++++++++
3 hours++++++++++++++++++++++++++++++++
6 hours+++++++++++++++++++++++++++++
24 hours-++++++++++++++++++++++++
2 days--+++++++++++++++++
5 days----+++++++++
8 days-------++++
Legend: - no growth;

+ - from 1 to 10 colonies;

++ from 11 to 30 colonies;

+++ from 31 to 100 colonies;

++++ - solid growth.

4
Table 3.

The results of studies of acute toxicity of tubiana on white mice.
The group of animalsDose, mg/kgThe number of miceNumber of mice:% death
deadsurvivors
1400606-
260062433
380065183
1000660100

Table 4.

The results of studies of acute toxicity of tubiana on white rats
The group of animalsDose, mg/kgThe number of ratsThe number of rats% death
deadsurvivors
14500606-
2500061516
3550063350
4600064266
5650065183
67000660100

Table 5.

The results of studies to determine the cumulative properties of tubiana
Duration of follow-up, days.Daily what about the input dose, mg/kgThe total dose for 4 days, mg/kgThe total dose, mg/kg
1-4283,7511351135
5-8425,61702,52837,5
9-12638,42553,65391,1
13-16957,63830,49221,4
17-201436,45745,614967
21-242154,68618,423585,4
253231,9-26617,3

Table 6.

Indicators embryotoxic activity tubiana
Indicators:The control groupExperienced group
The number of rats in the group55
The number of yellow bodies in the ovaries5351
Seats implantation5148
The number of live fetuses4945
The number of dead fetuses--
Preimplantational the death of zygotes, %of 3.77±0,7 5,9±0,23*
Postimplantation embryos death, %to 3.92±0,856,25±1,00
Total embryonic mortality, %7,55±0,9611,8±1,30
Fruit weight, g2,87±0,092,86±0,11*
Length plancey, cm1,29±0,011,28±0,01
Craniocaudal the fruit size, cm3,07±0,043,06±0,01*
Note:* - P<0,01

Table 7.

Change of body weight and duration of pregnancy in rats after intragastric administration of tubiana throughout pregnancy
Indicators:Groups of animals:
controlexperienced
The body weight of pregnant rats, g (% by source)107,2±0,7106,4±0,3*
on day 6, g121,7±0,9the 122.7±1,3
on the 15th day, g142,3±1,8144,7±2,1
The duration of pregnancy, DN23,2±0,223,0±0,4*

Table 8.

Indicators of the development of the offspring of white rats
Indices:Groups of animals:
controlexperienced
The number of pups born4846
Stillbirth, %0,2±0,20,4±0,2
Body weight of rats, g: neonates5,3±0,15,1±0,06*
on day 37,2±0,27,0±0,1*
Day 59,4±0,089,3±0,05
14th day16,8±0,316,6±0,08**
Day 2022,5±0,622,1±0,4
28-day31,6±0,529,3±0,8*
The term otlipaniya ears, days6,7±0,66,3±0,3
The period of pubescence, daysby 8.22±0,038,1±0,2
The term insight, days15,7±0,515,3±0,4
The period of eruption of the incisors, the days9,2±0,39,1±0,2
Postnatal mortality to 21 days, %of 4.44±0,66,6± 1,3*
Note:* - P<0,01, ** P<0,05

Table 9.

The test results chemoprophylactic activity tubiana and isoniazid in experimental tuberculosis of Guinea pigs.
Groups of animalsMedicationDaily dose, mg/kgExodusThe result of an allergic researchThe average index organs and Visavuori M.Average total index lesion and Visavuori M.
diedsurvivedBefore infectionAfter exposure throughPlace of infectionLymph nodesRegional limp. the nodeLightLiverSpleen
30 days60 days
ITurbofan20-8---0001/020/00/0 0/00/0
IITurbofan10-8---000/00/00/00/00/0
IIITurbofan5-8---000/00/00/00/00/0
IVTurbofan2,5-8-++0,8311,3/2,161,16/1,831,5/1,50,66/1,166,45/6,65
VTurbofan135-++1,32,83,3/3,163/2,83,5/3,53,16/317,06/12,46
VIIsoniazid10-8---000,37/00/00/00/00,37/0
VIIControl infected is s -4--++1,33,34,0/3,63,6/34/3,64/2,620,2/12,8
VIIIControl, intact--4---000/00/00/00/00/0
1 - the index of the organs;

2 - index Visavuori M.

1. Salt bis(oxymethyl)phosphinic acid with isonicotinic acid hydrazide (turbofan) of the formula I

2. The compound according to claim 1, with anti-TB activity.

3. A method of obtaining a compound according to claim 1, which consists in the interaction of bis(oxymethyl)phosphinic acid with isonicotinic acid hydrazide in methanol under stirring and heated to 60°C, followed by keeping at room temperature for 12-15 h and separation of precipitated sludge known techniques.



 

Same patents:

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to a method for preparing -amino-4-[hydroxy(methyl)phosphinyl]butyric acid of the formula (I): wherein R* means hydrogen atom or alkyl with 1-4 carbon atoms, or its salts with acid and bases. Method involves the following stages: a) (stage 1) trivalent methylphosphorus compound of the formula (II): wherein R1 and R2 mean independently halogen atom, (C1-C18)-alkoxy-, benzyloxy- or phenoxy-group that can be substituted, or one radical among R1 or R2 means hydroxy-group is subjected for interaction with unsaturated aldehyde or ketone of the formula (III): wherein R* has the abovementioned value in the presence of carboxylic acid anhydride and if necessary alcohols; b) (stage 2) the reaction mixture obtained at stage 1 is treated with ammonia/ammonium chloride and sodium cyanide, or with a mixture consisting of ammonia and hydrocyanic acid, or with a mixture consisting of ammonia and cyanide, if necessary, in the presence of ammonium chloride, and c) (stage 3) the reaction mixture obtained at stage 2 is hydrolyzed. The proposed method provides enhancing yield of compound of the formula (I) from 50% (by the known method) to 90%.

EFFECT: improved preparing method.

8 cl, 7 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention proposes (aminopropyl)methylphosphinic acids of the general formula (I): with exception of the following compounds: (1) (3-amino-2-hydroxypropyl)methylphosphinic acid racemate; (2) (S)-(3-amino-2-hydroxypropyl)methylphosphinic acid; (3) (R)-(3-amino-2-hydroxypropyl)methylphosphinic acid; (4) (3-amino-2-hydroxypropyl)difluoromethylphosphinic acid, and (5) (3-amino-2-oxopropyl)methylphosphinic acid showing affinity to one or more GABAe (γ-aminobutyric acid) receptor. Also, invention proposes their pharmaceutically acceptable salts, solvates and stereoisomers, and pharmaceutical compositions comprising indicated therapeutically active compounds and using indicated compounds for aims of therapy.

EFFECT: valuable medicinal properties of compounds and compositions.

12 cl, 15 ex

FIELD: chemistry of organophosphorus compounds, medicine, pharmacy.

SUBSTANCE: invention relates to new compounds of the formula (1) showing affinity to one or more GABAB receptors and their pharmaceutically acceptable salts, solvates and stereoisomers but with exception for racemate of (3-amino-2-hydroxypropyl)-phosphinic acid. Invention provides increasing the therapeutic index value.

EFFECT: improved and valuable properties of compounds.

14 cl, 1 tbl, 21 ex

The invention relates to derivatives of phosphinic and phosphonic acids of the formula (I)

where R1means unsubstituted or substituted phenyl, -O-(C1-C6)-alkyl, R2means hydrogen, RR3mean hydrogen, alkyl, unsubstituted or substituted phenyl, COOH group or - (CH2)2-CH(COOH)-NH-SO2-C6H4-C6H4-Cl(n), t stands for an integer of 1-4, And is a covalent bond, X is a group-CH=CH -, - group,- (CH2)about- where is 0,1,2 or 3, Y1and Y2mean-OH, -(C1-C4)-alkyl, -O-(C1-C4)-alkyl, and/or their stereoisomeric forms and/or physiologically acceptable salts

The invention relates to new compounds which can be used as inhibitors of matrix metalloprotease, in particular interstitial collagenases, and which is effective for the treatment of painful condition caused by excessive activity of matrix metalloprotease

The invention relates to heterocyclic chemistry and organo-phosphorus compounds, namely melamine salt of bis(oxymethyl)phosphinic acid of formula I (hereinafter referred to as Melafen") and how you can get it

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The specified connection is a regulator of growth and development of plants and may find application in agriculture and horticulture

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to new acid-additive nitrate salts of compounds taken among salbutamol, cetirizine, loratidine, terfenadine, emedastine, ketotifen, nedocromil, ambroxol, dextrometorphan, dextrorphan, isoniazide, erythromycin and pyrazinamide. Indicated salts can be used for treatment of pathology of respiratory system and elicit an anti-allergic, anti-asthmatic effect and can be used in ophthalmology also. Indicated salts have less adverse effect on cardiovascular and/or gastroenteric systems as compared with their non-salt analogues. Also, invention proposes pharmaceutical compositions for preparing medicinal agents for treatment of pathology of respiratory system and comprising above indicated salts or nitrate salts of metronidazol or aciclovir.

EFFECT: improved and valuable properties of compounds.

6 cl, 5 tbl, 19 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: the present innovation deals with identifying isoniazide, pyridine derivative, applied to control the quality of production manufactured by pharmaceutical enterprises and prepared by drug stores. One should detect by treating the sample under investigation with freshly prepared 10%-hydroxylamine hydrochloride alkaline solution at obtaining the color. The method creates minimal duration for detection.

EFFECT: higher sensitivity and specificity of the method.

1 ex, 2 tbl

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to new compounds representing bis-(3-guanidinopropyl)dodecylamine salts of the following formula: ,

wherein A means chlorine atom (Cl), HPO2-4, citrate, lactate, acetate, benzoate. These compounds can be used in preparing medicinal preparations for treatment of infectious diseases, in particular, tuberculosis and for preparing disinfectants for prevention of tuberculosis infection propagation.

EFFECT: valuable medicinal properties of compounds.

1 tbl, 1 dwg, 6 ex

FIELD: medicine, in particular vaccine of recombinant intracellular pathogens.

SUBSTANCE: invention relates to vaccine containing recombinant Calmette-Guerin bacillus (BCG), as well as method for prophylaxis of diseases associated with intracellular pathogen Mycobacterium tuberculosis. Disclosed is method for production of vaccine containing recombinant BCG having extrachromosomal DNA comprising gene encoding major Mycobacterium tuberculosis extracellular protein of 30 kD. Method for prophylaxis of mammalian diseases associated with intracellular pathogen Mycobacterium tuberculosis by administering of recombinant BCG expressing recombinant immunogenic antigens also is disclosed.

EFFECT: BCG of improved effectiveness.

6 cl, 4 ex, 7 tbl, 5 dwg

FIELD: medicine, phthisiology, pharmacy.

SUBSTANCE: invention proposes the inhalation preparation in treatment of tuberculosis comprising an antibacterial medicinal agent an aqueous solution. The preparation comprises also a medicinal preparation with the immunomodulating effect as 5-amino-2,3-dihydro-1,4-phthalazinedione potassium salt or 5-amino-2,3-dihydro-1,4-phthalazinedione sodium salt, or 5-amino-2,3-dihydro-1,4-phthalazinedione potassium and sodium salts taken in the equal ratio in the following ratio of components, wt.-%: antibacterial medicinal agent, 1.0-10.0; 5-amino-2,3-dihydro-1,4-phthalazinedione potassium salt or 5-amino-2,3-dihydro-1,4-phthalazinedione sodium salt, or 5-amino-2,3-dihydro-1,4-phthalazinedione potassium and sodium salts taken in the equal ratio, 1.0-10.0; water, the balance. The preparation inhibits activity of macrophages reducing the level of tumor necrosis factor (TNF) and acute-phase proteins that results to weakening intoxication symptoms, activates superoxide-forming function and phagocyte activity of neutrophile granulocytes, enhances the microbiocide system of cells that, in turn, results to arresting the inflammation process. Invention can be used as the inhalation agent in treatment of tuberculosis.

EFFECT: valuable medicinal properties of preparation.

2 cl, 2 ex

FIELD: medicine.

SUBSTANCE: one should perform the following stages: a) removal of contaminants out of plant; b) plant's reducing; c) treatment of reduced plant with laser radiation; d) suspending the mixture obtained at stage c) in water; e) maceration of suspension obtained at stage d) and f) separation of liquid developed. Composition should be obtained due to this technique. It should be applied at treating hepatitis C as an aqueous extract. It should be applied as aqueous extract as immunostimulant. Pharmaceutical preparation includes aqueous extract as an active constituent.

EFFECT: increased biological activity of the product.

43 cl, 16 ex, 1 tbl

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

FIELD: medicine, phthisiology, pharmaceutical industry.

SUBSTANCE: invention relates to applying betulin as an agent used in treatment and prophylaxis of tuberculosis, in particular, to applying birch bark as an agent for treatment and prophylaxis of tuberculosis in the content of betulin in birch bark extract above 70%. Betulin and birch bark extract promote to effective treatment and prophylaxis of tuberculosis. Invention can be used in treatment and prophylaxis of tuberculosis.

EFFECT: valuable medicinal properties of agent.

3 cl, 15 tbl, 6 dwg

FIELD: medicine, phthisiology.

SUBSTANCE: method involves administration of roncoleukin by lymphotropic route: subcutaneously, in region of pretracheal cellular tissue in the equal dose 1/4-1/5 of the average daily therapeutic dose, once in one or two days, 3 injections for a course. Invention provides the local delivery of roncoleukin to the injure zone directly that promotes to making depot of the latter in the injure focus and to the rapid elimination of pathogen from body of patients. Invention can be used for correction of immune insufficiency in patients with pulmonary tuberculosis.

EFFECT: improved method for correction.

2 ex

FIELD: medicine, phthisiology.

SUBSTANCE: additionally to chemotherapy including combination of antituberculous preparations based upon isoniazide or isoniazide and rifampicin one should prescribe zinc sulfate at daily dosage being 2.30-35 mg/kg due to twice or thrice oral intake during 21-28 d. The present innovation enables to shorten period of chemotherapy due to taking antilogarithms for the action of chemopreparations with zinc sulfate.

EFFECT: higher efficiency of therapy.

6 ex

FIELD: medicine.

SUBSTANCE: method involves rectally introducing mixture produced on base of 5% Novocain solution containing isoniazid, rifamycin, ethambutol in therapeutic doses daily during 21 days. The mixture is pretreated in ultrasonic field during 5 min at 2 MHz frequency.

EFFECT: improved bacteriostatic blood and prostate secret activity.

1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: the suggested composition includes therapeutically efficient quantity of para-aminosalicylic acid (PASA) as an active substance and target additives as sorbitol, polyvinyl pyrrolidone, salt of stearic acid, talc and citric acid. Pharmaceutical composition is designed as tablets covered with a membrane. The latter contains either "Acryl-iz" of Calarcon firm or composition consisting of copolymer of methacrylic acid with ethylacrylate, titanium dioxide, talc, propylene glycol and iron oxide. The suggested PASA-based antitubercular preparation meets all normative requirements of the State Pharmacopoeia, XI publication and has expiry terms of 2 yr.

EFFECT: higher efficiency of application.

6 cl, 3 ex, 1 tbl

FIELD: veterinary science.

SUBSTANCE: the suggested preparation contains Trichlorfon and 30%-ethanol at the following ratio of components, weight%: Trichlorfon 3.5-3.7 and 30%-ethanol or 3%-boric acid solution in 30%-ethanol - up to 100. Application of this innovation enables to shorten therapy terms and improves therapeutic effect.

EFFECT: higher efficiency of therapy.

4 ex

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