SU790622A1 - Sodium salt of n-4-nitro-phenylacetyl-tero-dl-3-nitrophenylserine having immunodepressive effect - Google Patents

Abstract

The sodium salt of n-4-nitrophenylacetyltreo-01-3-nitrophenylserine of the formula OH H I f ci - I H HN-e 0 μmg 2 02N has an immunodepressive effect.

Description

This invention relates to a novel chemical compound, the sodium salt of M-4-nitrophenylacetyltreo-1) L-3-nitrophenylserine, which has an immunosuppressive effect. This property makes it possible to assume its use in medicine. The closest compound in structure is the sodium salt of N-E6-phenylacetyl-Y-m-salicidene-threo-Bb-phenylserine, which has anti-inflammatory effect l. However, this compound does not have an immunosuppressive effect. The aim of the invention is to expand the arsenal of means of influencing a living organism. Known immunosuppressive agents, such as 6-mercaptopurine, azathioprine, 5-fluorouracil, the disadvantage of which is the undesirable side effect of 2j. This goal is achieved by a new chemical structure described by the formula // CC-COONa I / H HNCOCH. The compound of formula 1 is obtained by acylation of threo-DL-3-nitroserine with 4-nitrophenylacetic acid chloride in an aqueous alkaline medium, and the resulting N-4-nitrophenylacetyl-threo-Cb -3-nitrophenylserine is converted to the desired product in a methanolic solution of sodium methoxide. An example. To an ice-cooled solution of 4.52 g of 20.0 mol) Tpeo-DL-3-nitrophenylserine in 50 mp 0.4 n. caustic soda (20.0 mmol) in water with vigorous stirring at the same time for 10 minutes cold solutions of 4.0 g (20.0 mmol) of 4-nitrophenylacetic acid chloride in 10 ml of dioxane and 10 ml of 2 N are added dropwise. aqueous caustic soda (.20.0 mmol), following the fact that the reaction mixture remained weak alkaline during the entire time. the reaction mixture is stirred for another 1 h at room temperature / cooled with ice, acidified with 2 ml of concentrated hydrochloric acid. The isolated substances are filtered off, the filter is thoroughly rotted with water and recrystallized from a mixture of acetone and water (1: 1). Obtain 5.3 g (b8%) of N-4-nitrophenylacetyltreo-Cb-3-nitrophenylserine, m.p. 204-205 ° C (decomp.) The product is slightly yellowish crystals, insoluble in water, very weak in alcohol, soluble in dimethyl sulfoxide. IR spectrum (in tablets with KB on a Specord 71 IR instrument): C 0 stretching vibrations at 1645 cm and NH at 3270 cm in the amide group, simm. N0 stretching vibrations at 1345. UV spectrum (in ethyl alcohol on a Specord instrument (JV-U35) 203 nm (e 27100 lmol., "Ax 270 nm (15600 lmol G cm). Found: C 52.44; H 3, 88; N 10.79., Calculated,%: C 52.45; H 3.88; N 10; 79. To a solution of N-4-nitrophenylacetylTpeo-DL-3-nitrophenyl serine in an equivalent amount of 0.5N sodium methoxide in methanol, ether is added and the sodium salt is separated by N-4-nitrophenylacetyl-B-3-nitrophenyl serine, which is washed well with ether and dried in a vacuum desiccator (when melted it decomposes). T is 150-161 ° C. The product is amorphous powder with a grayish shade, well soluble in ode, methyl and ethyl alcohols, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, dimethylformamide, insoluble in ether, aliphatic and aromatic hydrocarbons, chloroform, vegetable oils. Biological tests show that the compound has a pronounced immunosuppressive effect. It is practically non-toxic. its LDso is 2500 mg / kg.) The biological activity of compound 1 is studied according to three tests characterizing the functional state of the immune system: 1) the effect on lyestransformation of lymphocytes itov; . 2) the effect on inhibition of game macrophages; 3) effect on the formation of hemaglutinins. Allergic action of the drug is studied using a skin allergy test with 2,4-dinitrochlorobenzene. The embryotropic action is studied indirectly (through the organism of mateia), by the effect of the preparation during the period of the organogenesis of the embryo. The study was conducted on CBA inbred mice, albino guinea pigs, Wistar rats, and peripheral human peripheral blood lymphocytes were used in the blast transformation reaction. Study of the effect of sodium salt of N-4-nitrophenyl-aceto-tpeo-DL-3-nitrophenylserine on lymphocyte blast transformation. Heparinized blood from healthy donors is used to formulate the lymphocyte formation blastran reaction (RTB). Mononuclear cells are isolated by centrifuging the blood in a ficoll-hypak gradient, the lymphocyte content is 40-60%. The number of viable cells determined by trypan blue staining is 90% and higher over the entire study period. Cells (1.5–10 ml) were cultured in medium 199 with the addition of antibiotics CHTO units / ml penicillin and 100 units / ml streptomycin), 10% of human AV-group syrup and 10 μg of PHA-P (Difco). At the time of post-culture culture, PHA was applied to the tested preparation in concentrations of 5, 10, 20, 30, and 60 µg / ml of culture medium. 4 hours before the end of the cultivation, 1 micron of N-thymidine was added to each tube (specific activity 17.6 Ci mM). After 72 h of the start of cultivation, each sample is transferred to millipore filters, washed with cold 10% trichloroacetic acid solution and saline. Radio activity is counted on a Mark 11 liquid scintillation counter. Results are expressed as percentages of a label relative to control stimulated cultures taken as 100%. The results of the experiment are given in .tabl. 1. From table. As can be seen in Figure 1, the drug tested in low concentrations of 5 to 10 µg / ml practically does not change the incorporation of the labeled precursor DNA compared to the control. At concentrations of 20, 30 and 60 µg / ml, the drug has an inhibitory effect, most pronounced at a concentration of 60 µg / ml (36% relative to the control). It should be noted that in the concentrations tested, the preparation does not have a cytotoxic effect. The incorporation of the labeled precursor into cultures of lymphocytes not stimulated with PHA after the introduction of the drug is the same as in control cultures without PHA. Study of the effect of sodium salt and 4-nitrophenylacetyltreo-01-3-phenylserine nitrate on macrophage migration inhibition. The method of inhibition of macrophage migration allows determination of the functional activity of T-effectors. Albino guinea pigs weighing 250-300 g are administered subcutaneously in a dose of 2 mg of BCG. Peritoxal macrophages from these animals are taken on the 21st day, after sensitization of BCG. The test drug is administered to BCG-sensitized guinea pigs at doses of 50 and 75 mg / kg, five times, starting from the day of sensitization. Two days before the reaction was initiated, 10 ml of sterile 2% peptone was intraperitoneally injected into the test and control guinea pigs. From the exudate of the abdominal cavity of these animals are cells. Cellular suspension of peritoneal exudate contains 25% polymorphonuclear leukocytes, 10% lymphocytes and 65% macrophages. The washed and resuspended cells in medium 199 are drawn into thin glass capillaries (diameter: 1.5 mm) and centrifuged at 800–1000 rpm in order to obtain a loose sediment of cells. Capillaries of the same length (1-1.2 cm) with cells are placed in special glass chambers and attached with heated paraffin. The chambers are filled with nutrient medium 199 containing 100 units / ml penicillin, 100 units / ml streptomycin and 10% inactivated normal guinea pig serum. Antigen tuberculin in a dose of 25 µg / ml, in a ratio of 0.8 MP medium 199 and O, 2 ml of antigen is added to the nutrient medium for the test samples. The edges of the chambers are smeared with sterile vaseline and covered with glass on top. Experimental and control chambers are incubated at 37 ° C for 24 hours, after which the chambers are placed under a microscope and using a drawing apparatus (PA-4), mounted on the microscope tube, the migration zones are drawn on paper, after which they are cut out , are weighed on a torsion balance, and calculate the percentage of inhibition of macrophage migration by the formula: migration area with antigen pmsdad migration without antigen The results of the experiments are shown in Table. 2. From table. 2 shows that the test drug inhibits the migration. - B1SC markofagov - sensitized sea pigs. in the presence of antigen. The inhibitory effect of the drug nabo.lee expressed in a dose of 75 mg / kg (almost 50% compared with the control, taken for 100%). The study of the effect of the sodium salt of I-4-nitrophenylacetyltreo-Bb-3-nitrophenylserine on humoral immunity. The experiments were carried out on inbred GULSH lines of the SLE using the method for determining hemagglutinin titers to sheep erythrocytes. The drug in doses of 50 and 75 mg / kg was administered to test animals intraperitoneally for three days, beginning on the day of immunization with sheep erythrocytes. Erythrocytes are administered intraperitoneally, once, at 0.3 mp of 50% suspension in saline. After 5 days, hemagglutinin titers are determined according to the generally accepted method. It was found that the sodium salt of N-4-nitrophenyl acetyl-threo-15L-3-nitro-phenyl-serine at doses of 75 mg / kg does not affect the hemagglutin titers that remain at the control level (1:88). nor the sodium salt of H-4-nitrophenylacetyl-trio-1) and-3-nitrophenylser1 on the allergenic effect in the dermal-allergic test with 2,4-dinitrochlorobenzene (2.4 DNHB). To determine the allergenic activity, the test drug is administered to experimental animals, then, using cutaneous application of 2.4 DNHB, the allergic contact dermatitis is induced and the effect of the drug on the development of the skin reaction to 2.4 DNHB is determined. If the preparation has an allergenic effect, then the reaction skin of 2.4 DNHB is enhanced, In the experiments, guinea pigs-albinos, males, weighing 250-300 g were used. 2 drops were applied to carefully trimmed skin of the right and left shoulder blades, 2-2 cm in size, using an eye-pipette. 50% solution 2.4 DNC, each drop of sensitizing dose is inserted into the skin with a glass rod. 2,4 DNBB was diluted with an alcohol-acetone mixture in a 1: 2 ratio. After 24-48 hours, an allergic contact dermatitis develops in guinea pigs. On . On the 14th day after sensitization, the animals are clipped on the back two areas of size cm each, and 1 drop of 2.4 DNHB is applied in the following concentrations: 1/250, 1/500 1/1000, 1/2500 - the resolving dose. If the test drug is sensitized by the body, then the reaction of 2.4 DNC is observed in high dilutions (1: 1000, 1: 25qO) N-4-nitrophenylacetyltreo-1 sodium salt L-3-nitrophenylserine is administered subcutaneously to guinea pigs a month before application, the sensitization of the dose with 2.4 DNC and continue to be injected. Diet drug to animals and in the period of developing allergic contact dermatitis, within two weeks. The drug was tested in the following doses: 10, 25, 50, 75 and 100 mg / kg. In each group there were 10 animals. The control, pigs applied with the skin 2.4 DNHB, the test drug was not administered. The intensity of the sensitization was assessed visually by the severity of the inflammatory reaction of the skin in place of the skin test by the tibal system: 0 - no visible reaction, 1 - weak erythema, 2 - clear erythema, 3- the same, with compaction, 4- cutting erythema with hemorrhage, pronounced infiltration, 5- serous-hemorrhagic cortex with ulceration. As a result of the experiments performed, it was found that the tested preparation in doses of 10, 25, 50, 75 and 100 mg / kg does not affect the course of allergic contact dermatitis induced by skin application of 2.4 DNCB. The intensity of the reaction in experimental animals is not significantly different from the control (0-1 point). Consequently, the test drug does not affect the development of the delayed-type hypersensitivity reaction. The study of the embryotropic action of the sodium salt of n-4-nitrophenylacetyl-trio-1) and-3-nitrophenyl-serine. The experiments were carried out on Wistar rats. The test drug was administered intramuscularly to animals from the 8th to the 15th day of pregnancy, which coincides with the period of organogenesis and placentation, in the following doses: 75 and 100 mg / kg. To reveal the effect of the drug on the fetus, animals are slaughtered on the 21st day of pregnancy. A necrosis of the uterus, ovaries, fetuses and placenta are examined at the autopsy. The number of yellow bodies in the ovaries, the number of implantation sites, the presence of live, dead and reserved fruits in the uterus are taken into account. On the basis of these data, pre-implantation and total embryonic gibel of fruits are calculated. All fruits and placenta are weighed and examined under a microscope MBS-1, while they note the presence of external changes and developmental disorders. The results of the experiments are presented in table. 3 As a result of the experiments, it was established that the administration of N-4-nitrophenylacetyl Tpeo-DL-3-nitrophenylserine sodium salt to animals at doses of 100 and 75 mg / kg does not cause an increase in total and post-implantational death of the fruit (P - 0.05). From these data, it can be concluded that the sodium salt of N-4-nitrophenylacetylTpeo-DL, -3-nitrophenylserine has an immunosuppressive effect, detected in cellular immunity models (reduces the incorporation of the labeled precursor DNA in lymphocyte cultures and inhibits the migration of macrophages) but does not affect humoral immunity (hemagglutinin titers in experimental animals at control level} The drug is not toxic with U-bso 2.5 g / kg. The effect of sodium salt H-4 on PHA-induced

Table of nitrophenylacetyltreo-g-3-nitrophenylserine lymphocyte blast-transformation 2 When testing new immunotropic drugs, it is extremely important to study the allergy and immunotropic effects of the test drug. The sodium salt of N-4-nitrophenylacetyltreo-DL-3-nitrophenylserine inhibits cellular immunity reactions, and a model of allergic contact dermatitis, which reflects the state of T-lymphocytes and is related to delayed-type hypersensitivity reactions, was selected for studying the allergic effect of this drug ( HRT). It is established that the compound of formula 1 does not possess allergenic and embryotoxic action.

Note. - In all experiments, standard errors are below 5-10%.

The effect of administering the sodium salt of H-4-nitrophenylacetyltreo-T) and-3-nitrophenylserine to inhibit the migration of markophages

Drug not

introduced

(control)

Table 2

100 Study of embryotropic action Tpeo-DL1 / 4 + 0.6

3.4 + 1.4 1.6 + 0.7 2.7 + 1.2 1.5 + 0.7 3.0 + 1.4 3, 6 + 0.04 0.61 + 0.01 3 , 5 + 0,005 0,59 + 0,003 3,95 + 0,03 0,63 + 0,01 Table 3 of sodium salt of N-4-itrophenyl acetyl 3-nitrophenyl serine

Claims (2)

Sodium salt of i-4-nitrophenylacetyl-Pb-3-nitrophenylserine formulas HE n g b-booka nkbobt! _g which has immunosuppressive effect. > 0 one 790622 2