Glutamylation complexes exhibiting immunostimulatory properties and method of production thereof

 

(57) Abstract:

The invention relates to new chemical substances with the General formula M'nM(H-1GluTrp)mn2Oh, where M' is a cation of an alkali metal, M is a cation d-metal or alkaline-earth metal, GluTrp - anion glutamylation, n is the number of atoms of alkali metal, m is the number of water molecules. The claimed compounds exhibit immunostimulating effect on a living organism. In the inventive solution provides a method of obtaining glutamylation of metal complexes, which is that glutamylation or its salt is subjected to interaction in the aquatic environment at 0-100oWith the equivalent amount of water-soluble salts of d-metal or alkaline-earth metal, or hydroxides, or oxides, or carbonates, or basic carbonates, followed by deposition of the target product with an organic solvent, and the interaction of the reactants and the precipitation of the desired product is carried out in the atmosphere, corresponding to the redox properties of d-metal. The technical result is achieved by expansion of the means that have immunostimulatory effects. 2 S. and 1 C.p. f-crystals, 5 PL.

The invention relates to new chemical ve the cation d-metal or alkaline-earth metal,

GluTrp - anion glutamylation,

n is the number of atoms of alkali metal,

m is the number of water molecules exhibiting immunostimulatory effects.

In addition, the invention relates to the field of production of new chemicals and can be used in the pharmaceutical industry.

A known number of coordination compounds of metals with bioligands with different specific biological effect.

For example, coordination compounds of bimetallic with histidine are antimasonry activity (ed. mon. The USSR 1438186).

The coordination compound of cadmium - bis-(geropotamos) cadmium pentahydrate - shows antiarrhythmic and hypotensive effect on a living organism (ed.mon. The USSR 1540548).

It is known that the connection of the dipeptide glutamylation H-L-Glu-L-Trp-OH - shows immunostimulatory effects, dosage form which is called "Timogen" [Trinus F. P. Pharmaco-therapeutic guide: 8th ed., - K.: Health, 1998. - S. 179]. But this tool is not actively.

The objective of the present invention to provide new chemical compounds by combining their composition and structures of monotonality funds.

In addition, the present invention is a method for these compounds, the set of operations and modes which are determined by the chemical nature of the claimed compounds.

The solution provided by the fact that the authors of the present invention synthesized glutamylation the metal complexes of General formula:

M'nM(H-1GluTrp)mn2OH,

where M' is a cation of an alkali metal,

M is a cation d-metal or alkaline-earth metal,

GluTrp - anion glutamylation,

n is the number of atoms of alkali metal,

m is the number of water molecules exhibiting immunostimulatory effects.

In addition, in the present invention it is proved that in the case when d-the metal is a cation of copper obtained compound has the most pronounced immunostimulating properties.

Then the problem is solved by the method of obtaining the above-mentioned compounds, which according to the invention in that glutamylation or its salt is subjected to interaction with water-soluble salt of d-metal or alkaline-earth metal or insoluble salts (carbonates, oxides, hydroxides in aqueous medium at t is aimogasta reactants and the precipitation of the desired product is carried out in the atmosphere, the corresponding redox properties of d-metal.

In addition, the invention anticipates receiving one of the stated compounds in which d-the metal is a cation of copper and which has the highest biological activity. It should be noted that in the case of cations of d-metals Mn(II), Co(II), Cr(II), Fe(II) atmosphere appropriate to their redox properties, is the atmosphere of inert gas, such as argon or nitrogen purged of oxygen.

The composition and structure of coordination compounds obtained were confirmed by the data of elemental analysis, thermogravimetry (PL.1), IR and electronic spectroscopy (PL.2, 3).

For evidence of the composition and structure of the claimed compounds were selected some of them, in particular, in which M - MP, Co, Ni, cu or Zn.

On the basis of the obtained data, the chemical structure of compounds may be expressed by this formula:

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We propose a method of obtaining glutamylation) complexes, namely, that glutamylation or its salt is subjected to interaction in the aquatic environment at 0-100oWith the metal salt or hydroxide, or oxide, or carbonate, or basic carbonate with subsequent deposition of a is menaut Sol element, belongs to the group of iron, salt of the platinum group metal salt and alkaline earth metal salt of a metal belonging to IB group of the Periodic system of elements, such as salt, copper salt of a metal which belongs to VIIa-group of the Periodic system, such as a manganese salt of a metal belonging to the IIB group of the Periodic system, for example zinc salts; usually applied chloride or sulfate of the above metals.

The yields of the target product is good and approximately 65-85%. The obtained substance are in crystalline form.

Compounds according to the invention have a broad spectrum immunostimulating action. They can be used in complex therapy of adults and children with acute and chronic infectious diseases, accompanied by a decline in cellular immunity, inhibition of reparative processes after severe injuries (broken bones), necrotic processes, as well as other conditions of immunodeficiency.

With the preventive purpose of coordination compounds of metals with glutamylation can be used to prevent the complications of infectious diseases, surgery, radiotherapy those whom they have a positive impact on the absorption capacity of neutrophils, expressed in phagocytic number, receptor the ability of neutrophils to the antigen of red blood cells E-ROP and to complement upon stimulation with PHA in the NBT-test. While known means timogen generally have less impact on these indicators and statistically changed only phagocytic number and the reserve capacities of neutrophils upon stimulation with PHA in the NBT-test.

Compounds according to the invention is used preferably interline or parenterally in a daily dose of 25 mg to 5 mg

The study of pharmacological activity of new glutamylation complexes was performed using a widely and successfully used in the present approach to study the action of pharmacological agents on the functional activity faguoqitirute cells (neutrophils) person in model experiments in vitro.

The choice of neutrophils as available, functioning of immune cells due to the fact that the neutrophil is a universal target and the metabolic indicator of immunological homeostasis in General. In turn, stimulated neutrophil itself can be a powerful effector and one of the trigger cascading RIT, different subpopulations of lymphocytes and other. The neutrophil provides suppression of natural killer cell reactions antialaising cellular cytotoxicity [Berezhnaya N. M. Neutrophils and immune homeostasis. - K.: Naukova Dumka, 1988, -187 C.]. The function of neutrophils the quenching of pathological (inflammatory process) related primarily to their elimination function, aimed at the destruction of destabilizing the immune and other biologically active systems factors. The neutrophil destroys chemoattractant produced by complement, kinny is responsible for the regulation of fibrinolysis, produces mediators that contribute to the change (rotation) of the cell phases in the pockets of pathology, thereby regenerative processes [Study of the blood system in clinical practice (Ed. by G. I. of Kozinets and C. A. Makarov). - M.: Triada - X, -480 S.; Dowdney C hematolog /A. F. Romanov, I. I. Vigovsky, B. C. Logsite that in. - K.: Health, 1997, - 342 S.; Hromycin N. Y., Kozlov Century A. Prostaglandins as a factor in the regulation of immune response // Immunology. - 1982. - 5. - S. 11-15]. Therefore, the condition of neutrophils may serve as an indicator of the nature and direction of the process in the direction of increasing or reverse development, as well as to assess possible effeciancy conditions neutrophils are in the inactive state, destroy unwanted tissue elements. Their functional activity and opportunities are revealed when exposed to a cell of various stimuli.

To assess the functional status of neutrophils (NF) blood generally accepted definition:

1. Their ability receptor - identification of receptors for antigen sheep erythrocytes (E - REF) and complement (EAC - ROP) method Jondal [Reznikov HP Complement and its role in immunological reactions. - M. : Medicine, 1967, - 247 S.].

2. Absorptive capacity is a measure of the percentage of phagocytosis (PF) and phagocytic number (FC). To perform this technique was evaluated phagocytic activity of neutrophils against laboratory strain of Staphylococcus 209 with the subsequent calculation of the values of PF and PC according to the standard technique [Chernushenko E. F., Kolosova HP Immunological research in the clinic., - K.: Health, 1978, - 179 C.].

3. Intracellular oxygendependent metabolism NF nitroimidazole (PCT) test is used to assess faguoqitirute ability of neutrophils in physiological conditions, but also under the influence of various factors [Duewa L. A., Cmut Century BC, Khilko T. F. Complex tests rosethorne for izucheniya and Prof. disease. -1982. - 9. - S. 24-27.; Gulling E. C. Forecasting the effectiveness of immunotherapy with levamisole sensitivity to it blood lymphocytes / Doctor/. case. - 1984. - 11. - S. 38-42].

In the formulation of model experiments in vitro to assess the functional activity faguoqitirute cells NF we have determined all of the above indicators with further calculating the magnitude of potential cells.

Experiment.

In a test tube containing phosphate-saline buffer, pH 7.4, in a total volume of 1 ml was made suspension NF 10 cells/ml. Then in the incubation medium were added to the study drug at the rate of 1 mcg/ml After careful mixing of the incubation mixture was placed in a thermostat, and incubated at 37oC for 30 minutes. After 30 minutes a sample was taken out and determined all of the above indicators. The results of the experimental samples were compared with those using the drug comparison thymogen in the same dosage. Table 4 shows the results of the study 5 drugs.

Preparations 1 and 2 did not cause statistically significant changes of the studied parameters, although the resulting increase in the values of indicators in the relative percentage of upon stimulation with PHA in the NBT-test) under the action of the drug 1 and increment values of the indicators in relative percent from 13.2 (receptor, the ability of neutrophils to the antigen of red blood cells E-REF) to 28.9 (phagocytic number, characterizing the adsorption capacity of neutrophils) performed studies are presented in tables 4 and 5. The 3 drug induced significant changes in phagocytic number, characterizing the adsorption capacity of neutrophils from 7.6 in the control (see table 5) to 11.9 in the experiment, the increase of the magnitude of the rate was 56.6% in relative interest (if control is taken as 100%). Also statistically significantly increased reserve capacity of neutrophils upon stimulation with PHA in the NBT-test with 10.1 (control) up to 14.4 in the experiment, the increase in the magnitude of the rate was 42.6% in the relative. Similar changes were obtained in the experiments with preparations 4 and 5. Preparation of 4 statistically changed 3 out of 5 of the studied indicators: phagocytic number, characterizing the adsorption capacity of the neutrophil receptor, the ability of neutrophils to the antigen of red blood cells E-RONG and reserve capacity of neutrophils upon stimulation in NBT-test. The drug 5 statistically changed 4 out of 5 of the studied parameters: the absorption capacity of neutrophils expressed phagocytic number, receptor the ability of neutrophils to the antigen of red blood cells E-ROP and to complement EAC-REF and backup capabilities it is ukazatelyam had "Timator" (NaCu(H-1GluTrp)3H2O). The results obtained in the experiments with the latter, are given in table 5. "Timator" on all studied parameters was superior to the comparator drug timogen (GluTrp), and two measures - the absorption capacity of neutrophils characterized by phagocytic number and backup capabilities of neutrophils, manifested upon stimulation with PHA in the NBT-test, changes were statistically significant. In all cases when compared with the control study indicators statistically significantly increased phagocytosis by 36.3 relative percentage of phagocytic number - 86,8 relative percent receptor, the ability of neutrophils to the antigen of red blood cells E-ROP and to complement EAC-RONF respectively 25.1 and 30,0 relative percent, and the reserve capacities of neutrophils, manifestiruuschem during stimulation in NBT-test, was increased by 68.6%. At the same time, the comparator drug timogen changed statistically only 2 test - phagocytic number - 35.5 relative percent and the reserve capacities of neutrophils upon stimulation with PHA in the NBT-test - 21.8 relative percent.

Thus, glutamylation complexes and especially the complex compound of copper (preparemodel and modification of molecules glutamylation due to the formation of coordination bonds with a metal ion. The invention is explained with specific examples of its implementation.

Example 1. 1,0031 g (of 0.003 mol) of glutamylation and an equivalent amount of metal salt (0,7179 g nSO44H2Oh, 0,7138 g l26N2Oh, 0,7134 g NiCl26H2O, 0,7486 g uSO45H2On or 0,8621 g ZnSO47H2O) separately from each other, each dissolved in 50 ml bidistilled water at room temperature. Solutions, if necessary, filtered and then immediately combine. To the solution was added two equivalents of alkali Paon (or KOH, LiOH), poured into 100 ml of anhydrous acetone, which is cooled with a mixture of ice and salt. Settles well otchaivaysya precipitate, which quickly sucked off on the cooled glass filter with a strong vacuum. The resulting material is dried in a vacuum desiccator over phosphoric anhydride. After repeated several times otsasyvanie substance slowly crystallizes and is then crushed into powder. Get the following outputs,%: MP-complex 75, 85 complex, Ni-complex 80, C-complex 80, Zn-complex of 70.

Similarly, when using an equivalent quantity of the appropriate mainly water-soluble metal salt can be obtained chap is vanadium. In the case of obtaining complexes of Mn (II), Co (II), Cr (II), Fe (II) reaction and filtration is carried out in the described conditions, but in an inert environment (the current of purified argon or purified from oxygen, nitrogen).

Example 2. 1,072 g of sodium salt of glutamylation and an equivalent amount of metal salt (0,7179 g MnSO44H2O, 0,7138 g l26N2Oh, 0,7134 g NiCl26H2O, 0,7486 g CuSO45H2O or 0,8621 g ZnSO47H2O) separately from each other, each dissolved in 50 ml bidistilled water at room temperature. Solutions, if necessary, filtered and then immediately combine. To the solution was added two equivalents of alkali Paon (or KOH, LiOH), poured into 100 ml of anhydrous acetone, which is cooled with a mixture of ice and salt. Settles well otchaivaysya precipitate, which quickly sucked off on the cooled glass filter with a strong vacuum. The resulting material is dried in a vacuum desiccator over phosphoric anhydride. After repeated several times the suction of the substance slowly crystallizes and is then crushed into powder. Get the following outputs, %: MP-complex 75, 85 complex, Ni-complex 80, C-complex 80, Zn-complex of 70.

Similarly when using the equiv is utiltarian complexes of palladium (II), platinum (II), magnesium, calcium, strontium, chromium (II, III), cobalt (III), vanadium. In the case of obtaining complexes of Mn (II), Co (II), Cr (II), Fe (II) reaction and filtration is carried out in the described conditions, but in an inert environment (the current of purified argon or purified from oxygen, nitrogen).

Example 3. To the freshly prepared metal hydroxides (in which the amount of metal the same as in 0,7179 g MnSO44H2O, 0,7138 g l26N2O, 0,7134 g NiCl26H2O, 0,7486 g CuSO45H2O or 0,8621 g ZnSO47H2O), obtained by standard methods, add an equivalent amount of glutamylation dissolved in 50 ml bidistilled water at room temperature. The resulting mixture was heated at 50-90oC in a water bath until complete restaurany hydroxide. The resulting solution was poured into 100 ml of anhydrous acetone, which is cooled with a mixture of ice and salt. Settles well otchaivaysya precipitate, which quickly sucked off on the cooled glass filter with a strong vacuum. The resulting material is dried in a vacuum desiccator over phosphoric anhydride. After repeated several times otsasyvanie substance slowly crystallizes and is then crushed into powder. Get the following output is whether the equivalent quantity of the appropriate mainly water-soluble metal salt can get glutamylation complexes of palladium (II), platinum (II), magnesium, calcium, strontium, chromium (II, III), cobalt (III), vanadium. In the case of obtaining complexes of Mn (II), Co (II), Cr (II), Fe(II) reaction and filtration is carried out in the described conditions, but in an inert environment (the current of purified argon or purified from oxygen, nitrogen).

Thus, it is proved that the claimed compounds exhibit immunostimulating effect on a living organism that transcends the known drug "Timogen". On the basis of the claimed compounds may receive medical and preventive preparatov with a wide range of immunostimulating action. The method of obtaining these compounds is quite simple in execution, which provides a high yield of the target product.

Example 4. 1,0031 g (or 0.003 mole) of L-glutamylation dissolved in 30 ml of bidistilled water and an equivalent amount of 0,2278 g (or 0.003 mole) of copper oxide CuO dissolved in 20 ml of a solution of hydrochloric acid containing 0,1095 g (or 0,006 mol) Hcl. The solutions are combined and, if necessary, the obtained solution is filtered. To the obtained solution under stirring add two equivalents of alkali, such as NaOH or KOH, LiOH), then poured into 100 ml of anhydrous acetone, cooled a chilled glass filter with a strong vacuum. The resulting material is dried in a vacuum desiccator over phosphoric anhydride. The dried substance is crushed to powder.

In the above operations receive the output of the synthesized substances, wt.%: C-complex 71.

Example 5. 1,0031 g (or 0.003 mole) of L-glutamylation dissolved in 30 ml of bidistilled water and an equivalent amount of 0,3562 g (or 0.003 mole) of carbon Nickel NiCO3when heated, dissolved in 20 ml of a solution of hydrochloric acid containing 0,1095 g (or 0,006 mol) Hcl. The solutions are combined and, if necessary, the obtained solution is filtered. To the obtained solution under stirring add two equivalents of alkali, such as NaOH or KOH, LiOH), then poured into 100 ml of anhydrous acetone, cooled with a mixture of ice and salt. When this deposited sediment, which is well defended. It immediately sucked off on a chilled glass filter with a strong vacuum. The resulting material is dried in a vacuum desiccator over phosphoric anhydride. The dried substance is crushed to powder.

In the above operations receive the output of the synthesized substances, mass %: Ni-complex - 74.

Example 6. 1,0031 g (or 0.003 mole) of L-glutamylation dissolve Vtoraya in 20 ml of hydrochloric acid, containing 0,1095 g (or 0,006 mol) Hcl. The solutions are combined and, if necessary, the obtained solution is filtered. To the obtained solution under stirring add two equivalents of alkali, such as NaOH or KOH, LiOH), then poured into 100 ml of anhydrous acetone, cooled with a mixture of ice and salt. When this deposited sediment, which is well defended. It immediately sucked off on a chilled glass filter with a strong vacuum. The resulting material is dried in a vacuum desiccator over phosphoric anhydride. The dried substance is crushed to powder.

In the above operations receive the output of the synthesized substances, wt.%: C-complex 70.

Example 7. 1,0031 g (or 0.003 mole) of L-glutamylation dissolved in 30 ml of bidistilled water and an equivalent amount of 0,6693 g (or 0.003 mole) of basic copper carbonate ISSS3Si(OH)2when heated, dissolved in 20 ml of a solution of hydrochloric acid containing 0,1095 g (or 0,006 mol) Hcl. The solutions are combined and, if necessary, the obtained solution is filtered. To the obtained solution under stirring add two equivalents of alkali, such as NaOH or KOH, LiOH), then poured into 100 ml of anhydrous acetone, cooled mixture denom glass filter with a strong vacuum. The resulting material is dried in a vacuum desiccator over phosphoric anhydride. The dried substance is crushed to powder.

In the above operations receive the output of the synthesized substances, wt.%: C-complex 71.

1. Glutamylation the metal complexes of General formula

M'nM(H-1GluTrp)mn2OH,

where M' is a cation of an alkali metal;

M is a cation d-metal or alkaline-earth metal;

GluTrp - anion glutamylation;

n is the number of atoms of alkali metal;

m is the number of molecules of water,

exhibiting immunostimulatory effects.

2. Glutamylation metal complex under item 1, characterized in that M is a cation of copper.

3. The method of obtaining glutamylation of metal complexes of General formula

M'nM(H-1GluTrp)mn2OH,

where M' is a cation of an alkali metal;

M is a cation d-metal or alkaline-earth metal;

GluTrp - anion glutamylation;

n is the number of atoms of alkali metal;

m is the number of molecules of water,

characterized in that glutamylation or its salt is subjected to interaction in the aquatic environment at 0-100oWith the equivalent amount of water-soluble salts of d-metal and the subsequent deposition of the target product with an organic solvent, thus the interaction of the reacting components and the deposition of the desired product is carried out in the atmosphere, corresponding to the redox properties of d-metal.

 

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