Complex 1:2 germany and 2,6-pyridinedicarboxylic acid, process for its production and pharmaceutical composition

 

(57) Abstract:

Describes the new germanium complex Germany and aromatic or non-aromatic, cyclic or heterocyclic 1,3-dicarboxylic acid, for example, bis(pyridine-2,6-dicarbonyl)Germany, has antibacterial interferoninducible and immunomoduliruushimi activity. Also describes the method of its production and pharmaceutical composition. 3 S. and 1 C.p. f-crystals, 4 PL.

The invention relates to antibacterial, immunomodulatory drugs, which can find application in medicine and veterinary.

The maintenance of homeostasis and resistance to the influence of environmental factors such as infectious agents, is determined to a large extent the operation of systems (primarily the immune system), providing the nonspecific resistance of the organism. The interferon system is one of the most important parts of nonspecific immunity. Interferons are cytokines, through which there are intracellular and humoral responses aimed at maintaining homeostasis of the body (Grsser, Cell Immunol., 977, 43, N 2, pp. 406-413; Stewart, The Interferon System, Springer-Verlag, N. Y., 1979; Soloviev and others, Interferon medicines the x elements, capable of producing various types of interferon, changing their functionality under the action of interferon and therefore realizing intracellular and humoral reactions, maintaining homeostasis of the organism. Defects in the interferon system lead to impaired function of the immune system and, consequently, to the increase in the risk of certain infections and cancers. Selective modulation of interferon inducers of interferons or other immunomodulators some parts of the immune system, directly involved in the protection against a particular disease, can lead to correction of the immunodeficiency and deficiency of interferon and significantly increase the body's resistance to infectious diseases (The Biology of the Interferon System. 1988. Proceedings of the Fifth Annual meeting of the International Society for Interferon Research (ISIR 88), Kyoto, Japan, 14-18 November, 1988, Tokyo, 1989, p. 503).

A new group of drugs - interferon inducers is widely used as drugs that suppress the infection, and as immunomodulatory drugs. Interferon inducers include heterogeneous groups of high and low molecular weight compounds of natural or synthetic origin. Such drugs is stimulating, radioprotective and so on (Ershov and others, Interferon and its inducers, M, Medicine, 1980, page 173).

Known preparations of alpha-, beta - and gamma-interferons, which, depending on the production method, are divided into natural (interferons first generation) and recombinant (interferons second generation):

1. Natural interferons: drugs-interferon - human leukocyte interferon, agitare, wellferon; drugs-interferon - human fibroblast interferon, Feron; -interferon - human immune interferon.

2. Recombinant interferons: drugs 2A-interferon IFN, readeron, Roferon; preparations 2b-interferon - intron, inrec; drugs 2C-interferon - berator; drugs-interferon - beta Feron; drugs-interferon - gamma-Feron (Chernev and other interferon System in normal and pathological condition, M, Medicine, 1966, pp. 196-221).

A significant disadvantage of natural interferon is the method of their derivation from human blood, which includes risk transfer heterogeneous genetic information and viral infections. Recombinant interferons do not have these disadvantages and are quite valuable in certain CL which supports their use. All the drugs interferon induce exogenous formation of interferon in the body, which is their common drawback.

Known inducers of interferon, used in clinical practice: synthetic compounds, such as, for example, Amiksin (small molecule compound of the aromatic series, belonging to the class of fluorenone), neovir (low-molecular compound related to biosdevname heteroaromatic compounds, to the class acridinone). Known natural compounds, such as, for example, magasin (the condensation product of hossipole through the aldehyde group with a sodium salt-aminoacyltrna acid), larigan (RNA phage with 2 double helix); Radostin (RNA double helix, obtained from a lysate of killer yeast Saccharomyces cerevisiae) (Ershov and other Antiviral drugs. St. Petersburg, 1993, page 104). The advantage of this group lies in its ability to induce autologous interferons. The above interferon inducers, however, induce the synthesis of alpha and beta interferons. Currently not known sufficiently effective inducers of IFN-γ, suitable for use in clinical practice.

It is known that germanium-organic Soini-sesquioxide General formula [GeCH2CH2COOH]2O3capable when administered orally to induce production of interferon in the blood of mice. Later it was shown that the drug is able to induce the synthesis of interferon in suspensions of mononuclear cells in vitro (Munakata et al, Interferon Res., 1987, v. 7, pp. 69-76). But it is toxic and has a low efficiency as inducer of immune interferon. Raw materials (germanium-chloroform) to obtain, there is enough.

Germanium complex of 2,6 - pyridinyl-bicarbonate (hydroxyprogesterone), having the properties of the inductor immune interferon, is described Ignatenko and others in the Russian author's certificate N 1622989 from 22.09.90. This substance, however, has a narrow range of immunomodulatory activity.

Thus, the aim of the present invention is to create a new drug that has anti-bacterial, non-interferon-inducing and immunomodulatory activity, which could be used in medicine and veterinary medicine for the prevention and treatment of infectious diseases, immunodeficiency and interferon-deficient state, and which would not have the disadvantages inherent in the known above-mentioned analogues.

According silicasol dicarboxylic acid, with two carboxyl groups are, respectively, 1 and 3 positions of the ring. The ring may have 0, 1 or 2 heteroatom, for example N, O or S. It can be 4-, 5-, 6 - or 7-membered. The specific embodiment of the present invention is bis (pyridine-2,6-in primary forms) Germany formula I:

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The new compounds possess antibacterial, interferon - inducing and immunomodulatory activities, which may find application in medicine and veterinary medicine for the prevention and treatment of infectious diseases, immunodeficiency and interferon - deficient state.

New complexes according to the invention can be obtained by reacting acid, such as 2,6-pyridinedicarboxylic acid, tetraalkoxysilane, preferably by tetramethoxysilane or tetraethoxysilane. Tetraalkoxysilane can be obtained directly in the reaction mixture, for example, from tetrachloride Germany and alcoholate of an alkali metal, for example, tetrachloride Germany and methanolate (ethanolate) sodium (obtained by dissolving metallic sodium in methanol (ethanol)).

The reaction may be carried out in a medium of an organic solvent, such as hexane, heptane, methanol or ethanolate inert gas, such as argon or nitrogen, the reaction time is about 3-5 hours Target product produce by conventional methods, for example, filtration and drying in vacuum, for example, when 133,322 PA (1 Torr) at 40oC.

The invention also relates to pharmaceutical compositions containing as active ingredient an effective amount of a complex of Germany together with a pharmaceutically acceptable carrier, excipient or diluent. The pharmaceutical composition can be administered in various ways, such as oral, parenteral, intranasal, etc.

In oral administration, the composition can be used in the form of tablets, aqueous suspensions, dispersible powders or granules, as well as in the form of syrups or elixirs. Compositions intended for oral administration can be obtained by any means known in this area used to produce pharmaceutical compositions. Such compositions may contain one or more agents selected from sweeteners, flavourings, colourings and preservatives used in pharmaceutical preparations with good aesthetic and taste.

Tablets may contain the active irrigatable tablets. These fillers can be neutral diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, granulating and loosening agents, for example, corn starch, derivatives of cellulose or alginic acid. Tablets may not have a shell or can be coated by known methods, for example, shell, delaying the process of disintegration of the tablet in the gastrointestinal tract, creating a prolonging effect. Aqueous suspensions may contain the active ingredient in a mixture with excipients suitable for the preparation of aqueous suspensions.

Pharmaceutical composition for parenteral administration, such as subcutaneous or intramuscular, may contain a sterile solution for injection in water or physiological solution. Intranasal pharmaceutical composition may contain a solution of the complex in Germany water or physiological solution.

As will be clear to experts in this field, the dose administered will depend on the desired effect, for example, immunomodulatory, interferon-inducing, anti-bacterial, as well as the severity of the disease, the age and condition of the patient. Usually, ADNOC complex has low toxicity. In studies of acute toxicity in mice LD50equal to 900 mg per kilogram of body weight.

Specific examples of making germanium-organic compounds of formula I, its physico-chemical characteristics, examples of pharmaceutical compositions and the results of biological tests is presented below.

The structure of the obtained compounds was confirmed by spectral analysis (IR, 1H and13C NMR, mass), the purity was confirmed by elemental analysis.

The IR spectrum of bis (pyridine-2,6-in primary forms) Germany received on the device "Bruker IFS 113v".1H NMR and13C NMR were obtained on a spectrometer Bruker AC-200". Mass spectrum (EU) was obtained on device "AT Finnigan Incos 50" at 70 eV.

Example 1.

6.4 g (of 0.038 mol) of 2,6-Pyridinedicarboxylic acid, 4.0 g (0.02 mol) tetramethoxysilane and 25 ml of absolute methanol under nitrogen atmosphere was heated under reflux with vigorous stirring at boiling point (64oC) within 3 hours the Precipitate was filtered Shott on a filter, washed with methanol (2x10 ml) and dried at a temperature of 40oC in vacuum (133,322 PA - 1 Torr) for 3 hours Output amounted to 6.88 g (90%) of the compound based on 2,6-Piri is allow with a melting point 303-305oC (decomposition). It is a medium soluble in water, diethylformamide and dimethylsulfoxide, and insoluble in hydrocarbons, chloroform, complex ether, ethanol.

IR (v, cm-1tablets at KBr): 667, 768, 922, 1095, 1148, 1304, 1368, 1493, 1600, 1732, 3097, 2800-3100.

1H NMR ( , memorial plaques, 200, 13 MHz, D2O): 8,35 (m, 6N, H-aromatic).

13C NMR ( , memorial plaques, 50,32 MHz, D2O): 129,7 ( -C, 143,2 ( - C), 147,8 ( -C), 167,4 (C=O).

Mass spectrum (m/z), (74Ge): 360 [M-CO2]+, 272, 228, 195, 171, 154, 139, 122, 112, 93, 84, 77.

Elemental analysis for C14H6GeN2O8:

Found,%: C 41,47; H 1,68; Ge 17,86; N 6,60.

Calculated,%: C The 41.75; H 1,50; Ge 18,02; N 6,95.

Molecular weight 402,76.

Example 2

To 1.1 g (0,048 mol) of sodium and 25 ml of absolute methanol at room temperature under nitrogen atmosphere was added 2.6 g (0.012 mol) tetrachlorethane within 5 minutes the Reaction mass was heated under reflux for 1.5 h, then was cooled to 20oC. the Precipitate (NaCl) was filtered Shott on the filter and washed with methanol (CH ml). To the obtained filtrate containing solution tetraethoxysilane in ethanol, was added 3 g (0.18 mol) of 2,6-pyridinedicarboxylic acid. Further processed as described in Example 1. The output amounted to 3.45 g (95%) in this way, essentially the same as described above in example 1.

Example 3

Solution for injection was prepared by dissolving bis(pyridine-2,6-in primary forms) Germany in water at room temperature. One ampoule of solution contains:

Bis(pyridine-2,6-in primary forms) Germany - 0.01 g

Water for injection 2 ml

Example 4

Tablets were prepared by grinding the ingredients in a mortar and the forming and pressing machine. One tablet contains:

Bis(pyridine-2,6-in primary forms) Germany - 25 mg

Sodium methylcellulose - 350 mg

Sucrose - 125 mg

The study of induction of interferon in mouse serum in vivo

Male CBA mice weighing 12-14 g were inoculated intraperitoneally compound I in the form of an aqueous solution in doses of 0.005-50 mg/kg Level synthesis of interferon in serum were determined at different intervals (5, 24, 48, 72 h). The interferon titration was performed in L-929 cell cultures. The test virus was the virus of mouse encephalomyocarditis. The results are presented in table 1.

As can be seen from table 1, compound I induces the production of serum interferon, the first 5 h after inoculation were identified interferons and , as in the last intervals-interferon. Optimation.

(a) a Cell culture of diploid human fibroblasts (M-19), highly sensitive to interferon, were seeded into wells of 96-well plates with medium Needle with 10% fetal calf serum. On the third day of growth, the culture was inoculated connection I in the amount of 1 μg/ml After 24-hour incubation (37oC, 5% CO2) was subjected to titration - interferon (IFN with an initial activity of 2106IE/ml vs. 100 TCD vesicular stomatitis virus). The susceptibility of cells to interferon was determined by the cytopathic effect of the virus. The results are presented in table 2.

(b) continuous Cell line J-41 with reduced sensitivity to interferon were seeded at a concentration of 105cells/ml in 96-well plate with medium 199 with 10% fetal calf serum. The next day was added to the culture of compound I in the amount of 1 μg/ml Titration of interferon and the sensitivity is carried out as described above in (a).

As can be seen from table 2, after 24-hour treatment of cells M-19 connection I, the sensitivity of cells to interferon alpha (IFN) has increased 8 times. After 24-hour treatment of cells J-41 connection I their sensitivity to interferon increased 4 race for the correction of interferon and enhance the sensitivity of patients to drugs interferon.

Research actions on hematopoietic stem cells

The connection I was diluted in saline at a concentration of 2.5 or 0.5 μg/ml Mice of the experimental group were inoculated intraperitoneally with 0.2 ml of one of the obtained solutions (0.5 and 1 μg/mouse, respectively). Control animals were treated with injections of saline. 1, 2, 3, 4, and 7 days after administration of the tested compound, the animals were injected heparin (40 units/mouse) and then took samples of peripheral blood, spleen and bone marrow from the femur. The blood was diluted 1:1 with saline. Suspensions of bone marrow and spleen were prepared by a conventional method. The content of hematopoietic stem cells (HSCs) in the blood, bone marrow and spleen of animals treated with the drug, was investigated by the method of exogenous koleeobrazovaniya in the spleen of lethally irradiated syngeneic recipients; more specifically, diluted peripheral blood (0.1 ml), bone marrow cells (3-5 104cells) or cells of the spleen (5105cells) were cultivated in lethally irradiated syngeneic recipients, which were scored on day 8-9; their spleen removed, fixed in a solution of Boina and counted the number of visible plaques or razlivochnogo marrow or spleen cells were treated in vitro with oximosilane in the amount of 1 mg/ml for 2 h at 37oC before inoculation of irradiated recipients. The number of HSC passed in S-phase was determined by the formula: A=(a-b) 100/a, where a is the number of HSC colonies without treatment oximosilane and b is the number of HSC colonies treated oximosilane. The results are presented in table 3.

As can be seen from table 3, the connection I increases the number of HSCs in the blood. It has been shown that there is no ability to stimulate the release of HSCs in S-phase.

The study of staphylococcal activity

The drug was inoculated subcutaneously to mice CBA in doses from 0.1 to 10 μg per mouse, after which the animal was inoculated 1,25 105microbial cells of Staphylococcus aureus Wood 46. Survival was defined as the percentage relative to the control group of mice that did not receive the compound I. the Results were, respectively, or 39.8%, 60.0% and 80.7% of at doses of 0.1, 1.0 and 10.0 μg. Thus, the dose of 1 μg increases the resistance of mice to Staphylococcus.

The study of influenza activity

The model system was experimental influenza infection in mice caused by laboratory strain WSN influenza virus A (H1N1). The virus has evolved over 9 days in chicken embryos. Mice C57B1/6 under the action of ether anesthesia were inoculated intranasally every 50 Icrowave. Control group animals received a placebo solution. Each group consisted of 10 male mice weighing 14-16, death of the animals was recorded every day for 14 days, and at the end of the experiment were defined as the average time of survival and mortality. The data presented in table 4.

The above data demonstrate the high influenza activity.

The study of clinical efficacy on dogs affected with distemper

Dogs with a diagnosis of distemper (disease duration from 10 to 30 days; 3 animals - intestinal form of the disease, in 2 - pneumonic and 3 animals neuroparalytic stage) were inoculated intramuscularly 2-4 ml (depends on weight of the animal) 0.5% solution of tested compound I in distilled water twice a day for 3-10 days. After this treatment, all the animals were recovered.

1. Complex 1:2 Germany and 2,6-pyridinedicarboxylic acid.

2. Complex under item 1, which is a bis(pyridine-2,6-in primary forms)Germany.

3. Pharmaceutical composition having antibacterial, interferon-inducing and immunomoduliruushimi activity, including an active ingredient and pharmaceutically reception is an effective amount of a complex of Germany under item 1.

4. The method of producing complex under item 1 or 2, which lies in the interaction tetramethoxysilane or tetraethoxysilane with 2,6-pyridinedicarboxylic acid.

 

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