RU2036930C1 - Peptides and composition on their base capable to regulate immune function in mammalian - Google Patents

Abstract

FIELD: organic chemistry. SUBSTANCE: peptides of the formula (I): Arg-Lys-Asp-Val-NR₁R₂ where NR₁R₂ - methylamide, 2-phenylhydrazine, piperidine amide, cyclohexylamide and isopropylamide, and composition containing compounds of the formula (I) and acceptable pharmaceutical vehicle. Compounds of the formula (I) were taken at concentration 0.001-100 mg/kg mass. Synthesized compounds were used in biology and medicine. EFFECT: improved method of synthesis. 5 cl, 1 tbl

Description

 The invention relates to peptides and compositions based on them, capable of regulating the immune function of mammals, which may find application in medicine.

 Immunomodulatory proteins, thymopoietin and tisplenin (or simply "splenin"), isolated from the goiter and spleen, respectively, of a bull and a person, are known. In addition, synthetic peptides are known in which the biological activity of thymopoietin and tisplenin has been further modified with the acquisition of additional qualitative features, such as resistance to enzymatic action.

 Known pentapeptidylopentin, which is an active site of thymopoietin and has the sequence Arp-Lys-Asp-Val-Tur. Peptide compositions are also known in which various substituents are present in the pentapeptide at the amino and / or carboxyl end. Both thymopoietin and thymopentin induce biological changes in two human T cell lines, CEM and MOLT-4, thereby showing their participation in stimulating T cell activity as a suppressor and as helper cells. The disadvantage of these peptides containing at least 5 amino acid residues in the peptide chain is their inaccessibility, in addition, they do not have the ability to stimulate the activity of the suppressor and helper of the human immune system in various T-cell disorders.

 It was first discovered that a number of tetrapeptides containing amides with terminal substitution of the carboxyl group have T-cell suppressor activity characteristic of thymopentin in cyclic GMP in CEM cells. This discovery is surprising in view of the fact that the same tetrapeptides with a free carboxy terminal group or amidated with various C-terminal amide groups do not cause such activity of the T-cell suppressor.

 The purpose of the invention is the search for a series of derivative peptides containing a small number of amino acid residues, new compounds with the ability to regulate insufficient or excessive T-cell function of the body, as well as the creation of compositions based on these peptides.

The problem is solved by peptides of the general formula I:
Arp-Lys-Asp-ValNR ¹ R ² where NR ₁ R _{2 is} selected from the group consisting of anilinamide, methylamide, 2-phenylhydrazine, piperidinamide, cyclohexylamide and isopropylamide; peptides having the ability to regulate the deficient or excessive T-cell function of an organism are preferred. As well as a composition capable of regulating the immune function of mammals, comprising a peptide and a pharmaceutically acceptable carrier, wherein said peptide is a compound of formula I:
Arp-Lys-Asp-ValNR ¹ R ² where NR ₁ R _{2 is} selected from the group consisting of anilinamide, methylamide, 2-phenylhydrazine, piperidinamide, cyclohexylamide and isopropylamide, in an amount of 0.001-100 mg / kg; a composition suitable for oral administration is preferred.

 Obtaining the peptide corresponding to the invention can be carried out by the method of solid-phase synthesis or standard methods of synthesis in solution (the method of solid-phase synthesis is a well-known and understandable method for producing peptides). The solid-phase synthesis method involves the phased introduction of protected amino acids into the growing peptide chain, which is linked by covalent bonds to a solid resin particle. In this procedure, by-products are removed by filtration, thus eliminating the need for purification of intermediate products. The generally accepted concept of this method depends on the connection of the first amino acid of the chain with a non-solid polymer by covalent bonds. Subsequent protected amino acids are administered singly or together in a phased manner until the desired sequence is formed. Finally, the protected peptide is removed from the solid resin base and the protecting groups are cleaved. Amino acids may be incorporated into any suitable polymer in the form of a resin. This resin must contain a functional group with which the first protected amino acid can be firmly bonded by covalent bonding. Various polymers are suitable for this purpose, such as cellulose, polyvinyl alcohol, polymethyl methacrylate, and N-alkylbenzylamine, N-alkylbenzhydrylamine and polystyrene resins. Suitable protecting groups used in such synthesis processes include tert-butyloxycarbonyl (BOC), benzyl (Bzl), tert-amyloxycarbonyl (AOC), tosyl (tos), ortho-bromophenylmethoxycarbonyl (BrZ), and phenylmethoxycarbonyl (Z or CBZ).

 In a general aspect, the peptide production procedure of the invention comprises the initial binding of a protected C-terminal amino acid to a resin. After this binding, the resin is filtered, washed and the protecting group (preferably BOC) at the alpha-amino group of the C-terminal amino acid is removed. Removal of this protecting group should be carried out without breaking the bond between the amino acid and the resin. The obtained resin peptide is then conjugated to a protected C-terminal penultimate amino acid. This conjugated binding is carried out with the formation of an amide bond between the free carboxyl group of the second amino acid and the amino group of the first amino acid bound to the resin. This sequence of procedures is repeated with subsequent amino acids until all amino acids bind to the resin. Finally, the protected peptide is cleaved from the resin in a conventional manner known to those skilled in the art, for example, the aminolysis method using cyclohexylamide, and the protecting groups are removed to form the desired peptide. The cleavage methods used to separate the peptide from the resin and to remove protecting groups depend on the choice of the resin and protecting groups and are already known to those skilled in the art of peptide synthesis.

 Peptides can also be obtained by standard methods of synthesis in solution, including stepwise or mass conjugate binding of amino acids or peptide fragments using chemical or enzyme methods for producing amide bonds. These solution synthesis methods are well known to those skilled in the art.

 The peptides of the invention exhibit a biological effect similar to that of human thymopentin. This biological activity is primarily determined by analysis with the measurement of inducing the formation of cyclic GMP in the human T cell line in comparison with human thymopentin. Inducing the formation of CMP by the peptide of the invention in this assay indicates the ability of this peptide to bind to the receptor site of human thymopentin on the cell and to induce biological activity similar to human thymopentin.

 Many of the described peptides have other very important advantages. Many of the peptides of the invention can be characterized by resistance to enzymatic degradation by both cleavage and serum enzymes. Thus, they exhibit a long half-life when administered by injection into a biological subject. Another advantage of many of these peptides is their ability to be orally administered. In view of the immunomodulatory characteristics of the claimed peptides, they find therapeutic application for treating people and possibly animals, since they have the ability to induce differentiation and maturation of T cells, which are able to participate in the body's immune response. As a result, the claimed peptides can be considered as peptides for multipurpose therapeutic use.

 The proposed peptides are able to impart general immunity to the body in that they increase or impart the ability to therapeutic stimulate cellular immunity. The claimed peptides or pharmaceutical compositions containing them find useful applications where cellular immunity is lost, and especially where there is an immunological deficiency. Thus, they are used to treat diseases associated with allergic reactions and autoimmune reactions. Where there is excessive antibody production, or cellular immunity caused by unbalanced T cells, the claimed peptides can correct this condition by stimulating the activity of the T cell suppressor. They find useful therapeutic use for some autoimmune diseases in which damaging antibodies are produced, such as lupus erythematosus, rheumatoid arthritis and others. In their broadest application, the described peptides or pharmaceutical compounds containing them are used to regulate the human or animal immune systems requiring such regulation. The term "regulation" means that the described compounds lead to a return of the immune system from an abnormal state, i.e. state of the disease, to a normal equilibrium state. Although such regulation can be widely used in the correction of immunological abnormalities, for example, Digueorg's syndrome, it can also be used to correct conditions of increased immunological activity, for example, autoimmune diseases. Thus, the invention encompasses methods for regulating the immune system of a human or animal in need of such regulation, which (methods) comprise the administration of an immunoregulatory-effective amount of at least one of the peptides into a human or animal organism, and also encompasses pharmaceutical compositions for implementing these ways. The invention encompasses a method of treating diseases caused by an absolute or complete deficiency of the body's immune system, especially in the function of a T-cell suppressor. The method comprises administering to the body a therapeutically effective amount of at least one of the peptides of this invention.

 The term "therapeutically effective amount" means the amount that is effective for the treatment of the above diseases. The invention also encompasses a method for inducing differentiation and maturation of T cells, which comprises administering to the body an effective inducing amount of at least one of the peptides of the invention. In addition, the invention encompasses pharmaceutical compositions for implementing these methods. To obtain pharmaceutical compositions that meet the invention, the proposed peptide is mixed as an active ingredient with a pharmaceutical carrier according to pharmaceutical methods of compounding. The carrier may take various forms, which depend on the form of the preparation desired for administration into the body, for example, for oral, sublingual, rectal, nasal or parenteral administration. In preparing the compositions in a preferred dosage form for oral administration, any commonly used pharmaceutical medium may be used. For the preparation of liquid preparations for oral administration, for example, suspensions, elixirs and solutions, a medium containing, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. may be used. For the preparation of solid oral preparations, for example powders, capsules, tablets, carriers such as starch, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. can be used. Formulations with controlled release may also be used. Because of their ease of administration, tablets and capsules are the most preferred oral dosage form, in which case solid pharmaceutical carriers are used. If desired, the tablets may be sugar coated or enteric coated by standard methods.

 For parenteral administration products, the carrier usually includes sterile water, although other ingredients that promote, for example, solubility or for protective purposes may be included. Injectable suspensions may also be prepared, in which case appropriate liquids, suspending agents, etc. may be used.

 The peptides of the invention are usually active when administered in amounts of about 1 g / kg body weight, and preferably in an amount of about 0.001-10 mg / kg body weight, although lower doses may be used. Typically, the same dosage range can be used to treat diseases or conditions mentioned above associated with immune deficiency. To suppress excess immune activity, higher dosage amounts are used, for example about 10-100 mg / kg body weight.

The following abbreviations are used to describe the examples: TFA trifluoroacetic acid; HOAc acetic acid; CH ₂ Cl ₂ methylene chloride; CH ₃ CN acetonitrile; DMF dimethylformamide; NH ₄ OAc ammonium acetate; n-BuOH n-butanol; pyr pyridine; DCC dicyclohexylcarbodiimide; HOBt 1-hydroxybenzotriazole; DMAR dimethylaminopyridine; TCA trichloroacetic acid; MeOH methanol; TLC thin layer chromatography; THF-tetrahydrofuran; EtOAc ethyl acetate; NaHCO ₃ sodium bicarbonate; MgSO ₄ magnesium sulfate; NMM N-methylmorpholine; Et ₂ O diethyl ether; HPLC high performance liquid chromatography; Pd / C palladium on carbon; DIEA diisopropylethylamine; iBuOCOCl isobutyl chloroformate; i-Pr ₂ O diisopropyl ether; ONp paranitrophenyl ester; RPMI tissue culture medium; PBS phosphate buffered saline.

 PRI me R 1. Synthesis of Arg-Pro-Asp-Val-isopropylamide.

2.25 g of BOC-Pro- (Bzl) Asp-Val are dissolved in 40 ml of CH ₂ Cl ₂ . The solution was cooled in an ice bath and 0.67 g of HOBt and 0.90 g of DCC in 4 ml of DMF were added thereto. After stirring for 10 minutes, 0.30 g of isopropylamine in 5 ml of CH ₂ Cl _{2 was} added. The mixture was stirred in an ice bath for 15 minutes, then warmed to room temperature. After 3 hours, the precipitate is filtered. The filtrate was extracted with 10% citric acid solution, water and saturated NaHCO ₃ solution. The organic layer was dried over MgSO ₄ and the solvent was distilled off in vacuo. The resulting product is an off-white solid, yield 2.32 g after crystallization from EtOAc-iPr ₂ O 1.64 g of product is obtained with a melting point 179,5-181,5 ° ^C.

To 0.84 g of the indicated product was added 50 ml of 40% TFA in CH ₂ Cl ₂ . The solution was stirred for 30 minutes, then the solvent was distilled off in vacuo. After introducing the ether into the residual distillation product, solid Pro (Bzl) Asp-Val-isopropylamide trifluoroacetate is formed. 0.93 g of BOC-arginine are dissolved in 10 ml of DMF. The solution was rapidly cooled to -20 ^{° C} and introduced into 18 ml of NMM, and then introduced 0.22 g iBu OCOCl. The mixture was stirred at −15 ^° C. for 20 minutes, then 0.18 ml of NMM and a cooled solution of trifluoroacetate in 10 ml of DMF were added. After 30 minutes, the cooling bath was removed and stirring continued for 2 hours. Most of the solvent was removed in vacuo. EtOAc and water are added to the residual product. These layers are separated and the organic layer is extracted with a solution of citric acid and a twice saturated solution of NaHCO ₃ . After removal of the solvent, 1.44 g of a colorless glassy substance remained. This product was purified by flash chromatography on silica gel 60, eluting with CH ₂ Cl ₂ : MeOH in a ratio of 98: 2. The first eluting component was the desired product. Pure fractions give 0.53 g of product. Then, 0.32 g of a product containing a small amount of impurities is obtained.

0.53 g of purified protected tetrapeptidamide is hydrogenated with 1 ml of formic acid in 20 ml of MeOH over palladium black. After one hour, the mixture is filtered through Celite and washed with water. MeOH is distilled off in vacuo. The residual distillation product is diluted with water, lyophilized and 280 g of product are obtained. This product is purified by chromatographic separation on P-Cefadex, eluting with 0.30 M NH ₄ OAc, pH 5. The fractions containing the center of the main peak are combined and lyophilized to give 190 mg of amorphous solid Arg-Pro-Asp-Val-isopropylamide .

 Amino acid analysis: Asp 1.01; PrO 1.03; Val 097; Arg 1.00.

Peptide Content 96%
Thin layer chromatography (Silica gel 60).

Rf (I) 0.44 (n-BuOH HOAc H ₂ O Ruz 15: 3: 12: 10)
Rf (II) 0.65 (EtOAc Purr HOAc H ₂ O 5: 5: 1: 3)
Rf (III) 0.16 (n-BuOH HOAc H ₂ O 3: 1: 1).

 PRI me R 2. Synthesis of Arg-Lys-Asp-Val-cyclohexylamide.

Into a solution of BOC- (Bzl) Asp-Val-ONp (1.09 g, 2.0 mmol) and cyclohexylamine (0.27 ml, 1.2 equiv) in THF (10 ml), HOBt (0.27 g) is added . The resulting yellow solution was stirred for 24 hours and evaporated in vacuo. The residual oil was diluted with EtOAc and washed with saturated NaHCO ₃ , H ₂ O, 10% citric acid and saturated saline. The organic phase is dried over MgSO ₄ , filtered, evaporated to give the protected dipeptidamide.

A protected dipeptide (0.71 g, 1.4 mol) is added to a saturated solution of 4.2 M HCl dioxane (10 ml). After 60 minutes, the reaction mixture was evaporated in vacuo to give a colorless solid as the hydrochloride salt of the dipeptide. In a mixed HCl solution of the dipeptide salt, (CBZ) ₃ Ary- (CBZ-Lys-ONp (1.34 g, 1.4 mmol) and HOBt (0.19 g, 1.0 eq) in DMF (10 ml), NMM (0.18 ml). A jelly-like precipitate forms after 16 hours. The reaction mixture is mixed with saturated NaHCO ₃ solution, filtered and washed with H ₂ O, 10% citric acid and H ₂ O and Et ₂ O. After recrystallization from 4 % HOAc / EtOAc receive (CBZ) ₃ -Arg- (CBZ) -Lys- (Bzl) -Asp-Val-cyclohexylamide as a colorless solid 1.36 g, 73%
This tetrapeptide (1.33 g) is deprotected in HF / anisole (30 ml / 8 ml) for 60 minutes at 0 ^° C. The residual product is mixed with Et ₂ O and filtered. The combined solids were dissolved in 10% HOAc and lyophilized to give 460 mg Arg-Lys-Asp-Val-cyclohexylamide.

This peptide is purified by high performance liquid chromatography (HPLC): Whatman M-20 10/50 ODS-3 column, flow rate 10.0 ml / min, 10% CH ₂ CN, 0.01 mol, pH 5 NH ₄ OAc. Three injection injections of 150 mg in 3 ml of buffer solution are carried out and the fractions eluted between 35.0 and 51.6 minutes are collected. After partial evaporation in vacuo and lyophilization, the tetrapeptidamide Arg-Lys-Asp-Val-cyclohexylamide is obtained as a colorless solid, 410 mg.

 Amino acid analysis: Arg 1.00; Lys 1.00; Asp 100; Val 0.85.

Thin layer chromatography (250 μm Silica gel G): Rf (I) 0.39 (n-BuOH: HOAc: H ₂ O: EtOAc 1: 1: 1: 1) Rf (II) 0.61 (n-BuOH: HOAc: H ₂ O: Pyr 15: 3: 12: 10)
Rf (III) 0.90 (EtOAc: HOAc: H ₂ O: Pyr 5: 1: 3: 5).

 PRI me R 3. Synthesis of Ary-Lys-Asp-Val-piperidinamide.

HOEt (0.68) is added to a mixed solution of BOC- (Bzl) -Asp-Val-ONp (2.72 g, 5.00 mmol) and piperidine (0.6 ml, 1.2 equiv) in THF (10 ml) g, 1.0 equiv.). After 48 hours, this yellow solution was evaporated in vacuo. The oily residual product is dissolved in EtOAc and washed once with saturated NaHCO ₃ solution, cold 2 M NaOH, twice with H ₂ O, once with 10% citric acid and saturated saline. The organic phase is dried with MgSO _{4 to} give a colorless oil BOC- (BZl) -Asp-Val-piperidinamide, 2.36 g.

After purification by flash chromatography with elution with MeOH / CH ₂ Cl ₂ , the protected dipeptidamide is obtained as a colorless solid, 1.65 g, 67%
In 4.2 M HCl-dioxane (10 ml), protected dipeptidamide (1.06 g, 2.17 mmol) is added. After 1 h, the solution was evaporated in vacuo, dissolved in CH ₂ Cl ₂ and evaporated again, and the result was an HCl salt in the form of a colorless oil.

Into a mixed solution of (CBZ ₃ -Arg- (CBZ) -Lys-OWp (2.08 g, 2.17 mmol), a colorless oil as described above, and HOBt (0.29 g) in DMF (10 ml) was added NMM (0.29 ml). After 1 h, a precipitate begins to form. After 16 hours, the reaction mixture is mixed with water. The product is extracted with EtOAc and washed with saturated NaHCO ₃ , H ₂ O, 10% citric acid, H ₂ O and Et ₂ O. Recrystallization from HOAc / EtOAc / Et ₂ O yields (CBZ) ₃ -Arg- (CBZ) -Lys- (Bzl) -Asp-Val-piperidinamide as a colorless solid, 2.40 g, 91%
The protected tetrapeptide (1.87 g, 1.55 mmol) is mixed in a nitrogen purged 10% HOAc (100 ml), to which 10% PdC (0.8 g) is added. The mixture is mixed in a Parr hydrogenator (500 ml vessel, pressure 43.5 psi-3 atm). After 48 hours, the reaction mixture was filtered through a 0.45 nylon filter, partially evaporated, diluted with H ₂ O and lyophilized to give the crude tetrapeptide as a colorless solid, 890 mg. This peptide is purified by HPLC (chromatography) column Whatman-M 10/50 ODS-3, flow rate 10.0 ml / min, 10% CH ₃ CN, 0.01 mol, pH 5 NH ₄ OAc. Three injections of 300 mg in 3 ml of buffer solution are carried out, and fractions eluted between 23.0 minutes and 44.0 minutes are collected. After partial evaporation in vacuo and lyophilization, the desired tetrapeptide amide Arg-Lys-Asp-Val-piperidinamide is obtained as a colorless solid, 510 mg.

Amino acid analysis: Arg 0.95; Lys 1.00; Asp 1.02; Val 1.00. Peptide Content 54.2%
Thin layer chromatography (25 microns, Silica gel G). Rf (I) 0.31 (n-BuOH: HOAc: H ₂ O: EtOAc 1: 1: 1: 1) Rf (II) 0.53 (n-BuOH: HOAc: H ₂ O: Pur 15: 3: 12:10) Rf (III) 0.61 (EtOAc: HOAc: H ₂ O: Pyr 5: 1: 3: 5).

 PRI me R 4. Synthesis of Arg-Pro-Asp-Val-N-methylamide.

Into a mixed solution of BOC-Val (10.86 g, 50.0 mmol) and DIFA (8.70 ml, 1.0 eq), methylamine HCl (3.38 g, 1.0 eq) and HOBT (7.66 g, 1.0 equiv.) in DC ₂ Cl ₂ and DMF, DCC (10.32 g, 50.0 mmol) was added at a ratio of 12: 5 (85 ml). A precipitate forms. After 2 hours, the reaction mixture was filtered and the filtrate was evaporated to dryness. The oily residue is dissolved in EOAc and washed three times with saturated NaHCO ₃ , H ₂ O, 10% citric acid and saturated saline. After drying on Na ₂ SO _{4, the} organic phase is filtered and evaporated, and the result is a colorless solid. Grinding it in hot hexanes gave the desired amide, BOC-Val-Methylamide, 7.98 g, 69% Melting point 120-122 ° ^C.

To this amide (4.60 g, 20.0 mmol) was added (50) TFA / CH ₂ Cl ₂ (20 ml). After stirring for 30 min, the solution was evaporated at less than 30 ^{° C,} the oily evaporation residue is dissolved in CH ₂ Cl _2, washed twice with saturated NaHCO ₃ solution. The organic phase is dried over Na ₂ SO ₄ , filtered and evaporated, and the result is Val-methylamide, 1.90 g, 73%
To a stirred solution of BOC-Bzl-Asp (4.72 g, 14.6 mmol), Val-methylamide (1.90 g, 14.6 mmol), HOBt (2.24 g, 1.0 equiv) in DMF ( 20 ml) DCC (3.01 g, 14.6 mmol) was added. After 5 minutes, a precipitate forms. After 16 hours, the reaction mixture was filtered and the filtrate was mixed with a semi-saturated NaHCO ₃ solution.

The resulting solids are combined, washed with H ₂ O, 10% citric acid, H ₂ O and Et ₂ O, dried with air and a colorless solid is obtained. Recrystallization from EtOAc gives the desired dipeptidamide, 5.85 g, 90% BOC- (Bzl) -Asp-Val-methyl-amide.

To this dipeptidamide (3.94 g, 9.05 mmol) was added 50% TFA / CH ₂ Cl ₂ (20 ml). After stirring for 30 min, the solution was evaporated at less than ^{30 °} C, and stirred with Et ₂ O, resulting in TFA dipeptide, 4.10 g, 100% as a colorless glass.

To a mixed solution of BOC-Pro (1.95 g, 0.95 mmol), TFA dipeptide (4.10 g, 9.05 mmol), DIEA (1.73 ml, 1.1 eq), DMA (0.12 g, 0.1 eq.) DCC (1.87 g, 9.05 mmol) was added. After 5 minutes, a precipitate forms. After 16 hours, the reaction mixture was filtered and the filtrate was mixed with a semi-saturated NaHCO ₃ solution. The resulting solid product is recovered, washed with H ₂ O, 10% citric acid, H ₂ O and Et ₂ O, air-dried to give the desired tripeptidamide, 4.15 g, 86%. This product is used without purification.

To this amide (4.15 g, 7.78 mmol) was added 50% TFa / CH ₂ Cl ₂ (15 ml). After stirring for 30 minutes, the solution was evaporated at a temperature lower than 30 ^° C, stirred with Et ₂ O and the result was a TFA tripeptide, 4.26 g, 100% as a colorless solid.

Into a mixed solution of 86.9 AOC-TOS-Arg (39.7 g, 7.78 mmol), tripeptidamide (4.25 g, 7.78 mmol), NMM (0.86 ml, 1.1 eq) and HOBt (1.19 g, 1.0 eq) in DMF (15 ml) was added DCC (1.61 g, 7.78 mmol). After 10 minutes, a precipitate forms. After 16 hours, the reaction mixture was filtered and the filtrate was mixed with a semi-saturated NaHCO ₃ solution. The resulting solid product is recovered, washed with H ₂ O, 10% citric acid, H ₂ O ether, dried with air and the result is a protected tetrapeptidamide, 4.82 g, 72%. Half of this solid product is deprived of protection in 50% TFA / CH ₂ Cl ₂ (10 mL) for 30 min, evaporated in vacuo and stirred with Et ₂ O. The resulting solid was cleaved by HF / anisole (30 ml / 8 ml) for 60 min at 0 ^C. The residue was stirred in EtOAc and extracted twice with 10% HOAc (50 ml). The aqueous extracts are lyophilized to give crude Arg-Pro-Asp-Val-methylamide.

This crude product is purified on an SPC-25 Sephadex (column 2.6 x 83 cm, 0.04-0.3 M NH ₄ OAc, pH 6.5; gradient: 100 ml / h (flow rate), 9.5 ml / fraction, 206 nm (detector) Fractions 113-128 are collected and lyophilized to give 925 mg of tetrapeptidamide, Arg-Pro-Asp-Val-methylamide.

 Amino acid analysis: Arg 1.06; Pro 0.99; Asp 0.95; Val 1.00.

Peptide Content 54%
Thin layer chromatography (250 microns, Silica gel G). Rf (I) 0.20 (n-BuOH: HOAc: H ₂ O: Upper phase 4: 1: 5) Rf (II) 0.26 (n-BuOH: HOAc: H ₂ O: Pur 15: 3: 12 : 10) Rf (III) 0.26 (EtOAc: HOAC: H ₂ O: Pyr 5: 1: 3: 5).

 PRI me R 5. Synthesis of N-formyl-Arg-Pro-Asp-Val-methylamide.

4.5 M HCl dioxane (10 ml) was added to AOC- (TOS) -Arg-Pro- (Bzl) -Asp-Val-methylamide (1.00 g, 1.17 mmol). After 60 min, the reaction mass is evaporated, diluted with H ₂ O and lyophilized, and the result is HCl Tetrapeptide in the form of a colorless solid powdery substance, 0.92 g, 100%
This solid was dissolved in DMF (10 ml) and DIFA (0.93 ml, 4.0 eq), DMAP (0.08 g) and para-nitrophenylformate (200 mg, 1.02 eq) were added. After 2 hours, the reaction mixture was treated with 10% citric acid. The resulting solid product is filtered, washed with H ₂ O, dried with air and the result is formylated peptidamide, 0.41 g

This solid was cleaved by HF / anisole (30 ml / 8 ml) for 60 min at 0 ^C. The residue was stirred in EtOAc and extracted twice with 1% NH ₄ OH (50 mL). The aqueous extracts are lyophilized to give a crude peptide, N-formyl-Arg-Pro-Asp-Val-methylamide. This crude product is purified on a DEAE Sephadex (column 2.6 x 90 cm, 0.1 M H ₄ HCO ₃ , non-buffer solution; flow rate 100 ml / h, 10 ml / fraction, 206 nm detector). Fractions 38-43 are collected, lyophilized and the result is an amidated peptide, 80 mg.

 Amino acid analysis: Arg 1.00; Pro 1.00; Asp 1.00; Val 1.00.

Peptide Content 50%
Thin layer chromatography 250 μm Silica gel G). Rf (I) 0.32 (n-BuOH: HOAc: H ₂ O: Upper phase 4: 1: 5)
Rf (II) 0.38 (EtOAc: HOAc: H ₂ O: Pyr 5: 1: 3: 5) Rf (III) 0.34 (trifluoroethanol: NH ₄ OH 1: 1).

 PRI me R 6. Synthesis of Arg-Lys-Asp-Val-2-phenylhydrazine.

To a stirred solution of BOC-Val (4,34 g, 20.0 mmol) in THF (40 mL) at ^-15 ° C is introduced NMM (2,30 ml) and then injected i-BuOCOCl (2,64 ml) . The resulting suspension was stirred for 15 minutes and then phenylhydrazine (1.98 ml) was added dropwise. The suspension is stirred at -10 ^to 0 ° C for 1 h and then warmed to room temperature. The reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The residual product is dissolved in EtOAc and washed with saturated NaHCO ₃ , H ₂ O and brine.

The organic phase is dried over Na ₂ SO ₄ , filtered, evaporated and the result is a solid orange substance. Recrystallization from CH ₂ Cl ₂ / petroleum ether gave BOC-Val-2-Phenylhydrazine as a colorless solid, melting point 140-141 ^{° C,} 4.96 g, 74%
BOC-Val-2-phenylhydrazine (1.13 g, 3.68 mmol) was added to a mixed solution of 4.2 M HCl of dioxane (10 ml). After 1 h, the reaction mixture was evaporated in vacuo, stirred in Et ₂ O and filtered, to give Val-2-phenylhydrazine in the form of a di-HCl salt, as a colorless solid, 1.03 g.

Into a mixed solution of this di-HCl salt (1.03 g, 3.68 mmol). BOC- (Bzl) -Asp (0.88 g) and HOBt (0.37 g) in THF (10 ml) were added NMM (0.60 ml) and then DCC (0.56 g). A precipitate forms almost immediately. After 16 hours, the reaction mixture was filtered and the filtrate was evaporated in vacuo. This residual evaporation product is dissolved in EtOAc, washed with saturated NaHCO ₃ solution and brine. The organic phase is dried over Na ₂ SO ₄ , filtered and evaporated in vacuo. After recrystallization from EtOAc / hexane, BOC- (Bzl) -Asp-Val-2-phenylhydrazine is obtained as a colorless solid, 1.07 g, 64%
Into a mixed solution of 4.2 M HCl of dioxane (10 ml), a BOC phenylhydrazide dipeptide (1.07 g, 1.75 mmol) is introduced. After 60 minutes, the solution was evaporated in vacuo to give a colorless oil, (Bzl) -Asp-Val-2-phenylhydrazide hydrochloride.

NMM (0.6 ml) was added to a mixed solution of (CBZ) ₃ -Arg- (CBZ) Lys-0 Np (1.68 g) of oil and HOBt (0.24 g) in DMF (6 ml). After 16 hours, a gelatinous precipitate forms. The reaction mixture is mixed with saturated NaHCO ₃ solution and the solids are removed, washed with H ₂ O, 10% citric acid, H ₂ O and Et ₂ O. After recrystallization from 2% HOAc / EtOAc, (CBZ) ₃ Ary- (CBZ ) -Lys- (Bzl) -Asp-Val-2-phenylhydrazide as a colorless solid, 2.15 g, 92% This protected peptide (1.88 g) is cleaved in HF / anisole (30 ml / 8 ml) in for 60 min at 0 ^C. The residual cleavage product is mixed with Et ₂ O and filtered. The solids were extracted with 10% HOAc (100 ml) for 1 h, filtered, the extract was lyophilized to give Arg-Lys-Asp-Val-2-phenylhydrazide as a colorless solid, 890 mg. This crude peptide is purified on a Sephadex CM (column 2.6 x 83 cm) 0.3 M NH ₄ OAc, non-buffer solution; flow rate 10 ml / h, 12.5 ml / fraction, 225 nm detector).

 Fractions 270-310 are collected, lyophilized and the result is 390 mg of the final product, Arg-Lys-Asp-Val-2-phenylhydrazide.

Amino acid analysis: Arg 0.98; Lys 1.03; Asp 1.00; Val 0.99. Peptide Content 83%
Thin layer chromatography: (250 μl Silica gel G) Rf (I) 0.39 (n-BuOH: HOAc: H ₂ O: EtOAc 1: 1: 1: 1) Rf (II) 0.51 (n-BuOH: HOAc: H ₂ O: Pyr 15: 3: 12: 10) Rf (III) 0.59 (EtOAc: HOAc: H ₂ O: Pyr 5: 1: 3: 5).

 PRI me R 7. Synthesis of Arg-Lys-Asp-Val-aniline amide.

Into a mixed solution of BOC-Val (4.35 g, 20.0 mmol), aniline (3.0 ml, 1.05 equiv) and HOBt (3.09 g, 1.0 equiv) in DMF (20 ml) at DCC (4.12 g, 20.0 mmol) was added at room temperature. A thick precipitate forms within 5 minutes. After 4 hours, the reaction mixture was filtered and the filtrate was poured into semi-saturated NaHCO ₃ (50 ml). The resulting precipitate is recovered. The solids are washed with H ₂ O (1: 1) and dried in air. Recrystallization from hexane-isopropanol gives a colorless solid product, anilide BOC-Val-aniline amide, 3.76 g, 64%
This BOC-Val-anilinamide (2.50 g, 8.55 mmol) was dissolved in 50% TFA / CH ₂ Cl ₂ (10 ml) and stirred for 30 minutes. The reaction mixture was evaporated in vacuo at room temperature, and the residual evaporation product was dissolved in CH ₂ Cl ₂ . This solution is washed twice with a saturated solution of NaHCO ₃ , dried over MgSO ₄ , filtered and evaporated, and the result is Val-anilinamide as a pale yellow oil, 1.42 g, 88%
To a solution of BOC- (Bzl) -Asp (2.38 g, 1.0 equiv), Val-anilinamide and HOBt (1.13 g) in DMF (7.5 ml), DCC (1.52 g, 1, 0 equiv). A precipitate formed and the reaction mixture was stirred for 3 hours. The mixture was filtered, the filtrate was mixed with saturated NaHCO ₃ solution. The resulting solids are recovered, washed with H ₂ O and air dried. Recrystallization from EtOAc / petroleum ether gave the colorless BOC-dipeptide amide, mp 121-122 ^{° C,} 1.74 g, 46% BOC- (Bzl) -Asp-Val - Anilineamide.

The BOC dipeptide (1.71 g, 3.44 mmol) is dissolved in 50 TFA / CH ₂ Cl ₂ (10 ml) and stirred for 30 minutes. The reaction mixture is evaporated in vacuo at room temperature and the residual product is stirred in ether, filtered and air dried.

To a stirred solution of BOC- (Cbz) -Lys (1,33 g, 3.50 mmol) in DMF (10 mL) at -20 ^{° C} is introduced NMM (0,46 mL, 1.2 eq) and then i- BuOCOCI (0.46 ml, 1.01 eq). The resulting suspension is stirred for 30 minutes and then the TFA salt of the dipeptide is added, followed by NMM (0.46 ml). The reaction mixture is stirred and warmed to room temperature. After 1 h, the reaction mixture was poured into a semi-saturated NaHCO ₃ solution (20 ml). A colorless solid forms. This substance is extracted and dried by air. Recrystallization from EtOAc / i-Pr ₂ O is obtained BOC tripeptide amide, 2.05 g, 78% Melting point 164-167 ° ^C.

BOC tripeptidamide (2.00 g, 2.63 mmol) was dissolved in 50% TFA / CH ₂ Cl ₂ (6 ml) and stirred for 30 minutes. The reaction mixture was evaporated at room temperature in vacuo, and the evaporation residue was crushed in ether, filtered and air dried to give the TFA salt.

To a stirred solution of (CBZ) ₃ Arg (1,52 g, 2.63 mmol) in DMF at -20 ^{° C} is introduced NMM (0,35 mL, 1.2 eq) followed by i-BuOCOCl (0,35 ml 1.01 eq). The resulting suspension was stirred for 30 minutes. Then, NMM (0.35 ml) was added, followed by the TFA salt. The reaction mixture was warmed to room temperature for 1 hour. The mixture was poured into a saturated NaHCO ₃ solution and the resulting solids were filtered, washed with H ₂ O and air dried. After stirring in EtOH, a protected tetrapeptide, BOC- (CBZ) -Lys- (BZl) -Asp-Val-anilinamide, 2.55 g, 78% is obtained
Palladium black (0.95 g) is introduced into a variable suspension of tetrapeptide (2.30 g, 1.89 mmol) in trifluoroethanol (50 ml) and formic acid (97% 5 ml). The reaction mixture is stirred vigorously. After 1 h, the solution was filtered through Celite and washed with H ₂ O. The filtrate was partially evaporated in vacuo, the residual product was lyophilized, and a colorless solid was obtained, 0.89 g.

The crude peptide was purified on a SPC-25 Sephadex (column 2.6 x 89 cm, 0.1-0.3 mol NH ₄ OAc; pH 5 gradient; flow rate 100 ml / h, 15 ml / fraction, 206 nm detector). Fraction 253-285 was collected, lyophilized and 680 mg of Arg-Leu-Asp-Val-anilinamide was obtained.

 Amino acid analysis: Arg 1.00; Lys 0.98; Asp 1.00; Val 1.01.

Peptide Content 100%
Thin layer chromatography (250 μl Silica gel G) Rf (I) 0.42 (EtOAc: HOAc: H ₂ O: Pyr) (5: 1: 3: 5) Rf (II) 0.32 (nBuOH: HOAc: H ₂ O : Et0Ac) (1: 1: 1: 1) Rf (III) 0.54 (nBuOH: HOAc: H ₂ O: Pyr) (15: 3: 12: 1)
PRI me R 8. Synthesis of Arg-Lys-Asp-Val-methylamide.

 A. Na-tert-butyloxycarbonyl- (N-benzyloxycarbonyl) lysyl- (β-benzyl) -partyl-valine-methylamide.

3.21 g of Na-tert are dissolved in 50 ml of 85% acetic acid. butyloxycarbonyl- (N-benzyloxycarbonyl (lysyl- (β-benzyl) aspartyl) -valyl-penacyl ester. Zinc powder (5.2 g) was added and the mixture was stirred vigorously for one hour. The reaction mixture was filtered, the filtrate was diluted with water and extracted three times with ethyl acetate. The combined organic layers are extracted with water and a saturated aqueous solution of sodium chloride, then dried over anhydrous magnesium sulfate. After removal of the solvent, oil remains. Ether is introduced, then hexane and a gum are formed minutes a precipitate. This product is mixed with the trituration with hexane until it turns until a dry white solid in an amount of 1.75 This product is dissolved in 14 ml of ethyl acetate and 1 ml dimethylformamide. After cooling the solution to ^-20 ° C is injected 0 30 ml of N-methylmorpholine, then added dropwise 0.36 g of isobutyl chloroformate. The reaction mixture was stirred at -15 ^{° C} for 20 min, then injected 0.29 ml N-methylmorpholine and a chilled solution of 0.18 g methylamine hydrochloride in 10 ml of 95% ethanol. A thick precipitate forms. To speed stirring introduced 5 ml of dimethylformamide and the mixture was stirred at ⁰ ° C for 90 min, then at room temperature for 30 min. The reaction mixture is introduced into 75 ml of water. The organic phase with stirring of the mixture turns into globules of a waxy solid. This solid is filtered and the filtrate is extracted with ethyl acetate. The residual evaporation product of the organic phase is combined with this solid and crystallized from ethyl acetate-isopropyl ether. The product yield is 1.53 g, melting point 183.5-184.5 ^about C.

 Elemental analysis data: C 61.67 (61.96); H, 7.21 (7.37); N, 9.81 (10.04).

B. Tri-benzyloxycarbonyl-arginyl- (N-benzyloxycarbonyl) -lysyl- (β-benzyl) Asp-artyl-valine-methylamide
50 ml of 50% trifluoroacetic acid in methylene chloride are introduced into 1.46 g of the product from section A. After stirring for 25 minutes, the solvent is evaporated, ether is introduced into the residual evaporation product, and a white solid is obtained. This product is filtered and washed with ether.

Tribenzyloxycarbonyl-arginine (1.33 g) is dissolved in 10 ml of DMF. The solution was cooled to ^-20 ° C and injected 0.25 ml of N-methyl morpholine followed by addition of 0.31 g isobutylchloroformate in 2 ml DMF. The mixture was stirred at -20 ^{° C} for 20 min, then injected 0.26 ml N-methylmorpholine and cooled tripeptidtriftoratsetata dissolved in 10 ml of DMF. The reaction mixture was stirred at a temperature of (-20) - (- 10) ^C for 30 min, then at room temperature overnight. After 16 hours, the mixture is introduced into 125 ml of water. The precipitate is recovered by filtration and washed with warm methanol and ethyl acetate. The product yield is 1.74 g, melting point 118-120 ^about C.

Amino acid analysis: Asp 0.99; Val 1.03; Lys 0.98; Arg 1.00; peptide content 67.7%
C. Arninyl-lysyl-aspartyl-valine-methylamide
1.72 g of the product from section B undergoes reduction of 2 ml of formic acid over 1 g of palladium black in 40 ml of trifluoroethanol in order to remove protection. The mixture is rapidly mixed for one and a half hours in a narrow-necked flask. Palladium is removed by filtration through Celite with washing in water. Most of the trifluoroethanol is removed from the filtrate in a rotary evaporator. The residual product is diluted with 5% acetic acid and lyophilized. The yield of 676 mg.

The peptide is purified by chromatographic separation in a column of SP-Sephadex 2.6 x 85 cm with an elution gradient of 0.20-0.50 N. NH ₄ OAc, pH 5.5, 3 L. Fractions of 10 ml are collected, control is carried out at 206 nm. The main peak is detected in fractions 185-220. Three fractions were selected: 185-190, 191-205, 206-220. Lyophilization yields yields of 21 mg, 497 mg, and 235 mg, respectively. The first fraction contains an impurity, the other two fractions have a degree of purity of more than 99.5%
High Resolution Liquid Chromatography Analysis, M. Bondapak C ₁₂ : rt 8.5 min with 0.01 M NH ₄ OAc, pH 5 at 2.0 ml / min.

Amino acid analysis: Asp 1.01; Val 0.95; Lys 1.01; Arg 1.02; peptide content 56%
Thin layer chromatography (Silica gel 60) Rf (I) 0.19 (n-BuOH: HOAc: H ₂ O: pyridine 15: 3: 12:10). Rf (II) 0.15 (EtOAc: Pyr, H ₂ O 5: 5: 1: 3) Rf (III) 0.45 (TFA: NH ₄ OH 2: 1).

 PRI me R 9. Synthesis of Arg-Lys-Asp-Val-2-propylamide.

A. α-BOC- (CBZ) -Lys- (β-Bzl) -Asp-Val
Into a rapidly mixed solution of α-BOC- (CBZ) -Lys- (β-Bzl) -Asp-Val-phenacyl ester (8.02 g, 10.0 mmol) in 85% HOAc (100 ml) at room temperature zinc etched acid (12.4 g) was introduced at an acid etched temperature. After 1 h, the reaction mixture was filtered and the filtrate was evaporated in vacuo. The oily residue was stirred with trituration in Et ₂ O. The resulting solid was recovered, washed with Et ₂ O and dried in vacuo at 40 ^C to yield 4.95 g, 72%
B. α-BOC- (Cbz) Lys- (β-Bzl) -Asp-Val-NHCH (CH ₃ ) ₂ .

To a stirred solution of BOC tripeptide (1.00 g, 1.46 mmol) in THF (5 mL) at ^-15 ° C is introduced NMM (170 ul, 1.06 eq) and OSOSl (195 .mu.l, 1.03 eq) . The resulting slightly turbid solution was stirred for 15 minutes and then 2-propylamine (150 μl, 1.1 equiv) was added. The reaction mixture was warmed to -5 ^C. The gelatinous solid is formed and the reaction mixture was warmed to room temperature. After 16 h, the reaction mixture was evaporated in vacuo at ³⁰ ° C and the solid residue was washed with 10% citric acid, H ₂ O, saturated NaHCO ₃ and H ₂ O. After air drying gave the colorless BOC tripeptide amide, 2.58 g, 55%
C. (Cbz) ₃ Arg- (Cbz) -Lys- (β-Bzl) -Val-NHCH (CH ₃ ) ₂ .

BOC tripeptidamide (0.55 g, 0.76 mmol) was dissolved in 4.4 HCl of dioxane (5 ml) and stirred for 45 minutes. The solution was evaporated in vacuo and the solid residue was triturated with Et ₂ O. After filtration, a colorless HCl tripeptidamide 0.51 g, 100% HCl solution of tripeptidamide, (Cbz) ₃ -Arg-para-nitrophenyl ester (0.53 g) was obtained. 1.0 eq) and HOBT (0.12 n, 1.0 eq) in DMF (5 ml) at room temperature, NMM (92 μl, 1.1 eq) was added. Slow sedimentation occurs for 16 hours. The gelatinous reaction mixture is stirred with saturated NaHCO ₃ solution and filtered. The solid precipitate was washed with H ₂ O, 10% citric acid, H ₂ O and Et ₂ O. The obtained protected tetrapeptidamide was recrystallized from HOAc / EtOAc to give a colorless solid, 743 mg, 83%
D. Arg-Lys-Asp-Val-NHCH (CH ₃ ) ₂
The protected tetrapeptide (0.70 g, 0.59 mmol) is suspended in 90% HOAc (50 ml). The mixture was purged with N ₂ and treated with 10% Pd / C (0.4 g). After hydrogenation in a Parra shake apparatus (250 ml vessel, P _o 52.0 psi 3.64 atm) for 64 hours, filtration through a 0.45 μm nylon filter, partial evaporation and lyophilization, a crude tetrapeptide, 385 mg, is obtained.

The crude peptide was purified on Sephadex CM (column 2.6 x 93 cm, 1.7 L, 0.2 mol, then 0.3 mol, NH ₄ OAc, non-buffered solution; flow rate 100 ml / h, 10 ml / fraction, 225 nm detector). Fractions 335-370 merge and lyophilize to give 231 mg of product.

Amino acid analysis: Arg 1.00; Lys 1.02; Asp 0.98; Val 1.00; peptide content 80.3%
Thin layer chromatography (250 μm, Silica gel G) Rf (I) 0.24 (n-BuOH: HOAc: H ₂ O: EtOAc 1: 1: 1: 1) Rf (II) 0.52 (n-BuOH: HOAc: H ₂ O: pyridine 15: 3: 12: 10)
Rf (III) 0.61 (EtOAc: HOAc: H ₂ O: pyridine 5: 1: 3: 5).

 Biological activity cyclic GMP analysis.

 This assay determines the ability of a peptide of the invention to bind to a cell membrane receptor of a CEM cell and selectively stimulate the formation of cyclic CMP like human thymopentin.

 The CEM cell line was obtained from the American Type Culture Collection, Rockville, Md. CEM cells were seeded, grown for 3 days and harvested.

Cells were washed three times in PBS and resuspended to a concentration of 1640 RRMI 1,0h10 ⁷ cells / ml and equilibrated at 37 ^{° C} for 30 min and then administered the test peptides (25 ul) and control peptides. The incubation was carried out in a shaken water bath for 4-5 minutes and then the process was stopped by introducing 1 ml of ice-cold TCA (10%).

Cells in TCA were homogenized and sonicated to isolate the cyclic nucleotide. This suspension is centrifuged at 3000 x g for 20 min at 4 ^C. The resulting precipitate was dissolved in 0.1N. NaOH to determine protein content. TCA was removed from the surface fraction by extracting 4 times with 5 ml of water-saturated Et ₂ O. After the final extraction, the remaining traces of ether were removed by heating for 10 minutes in a water bath at 50 ^C. After lyophilization the sample was injected in 50 mM acetate buffer solution (pH 6.2) for cyclic CMP radioimmunoassay.

 For example, a dose-response curve was determined for each compound using 1000 μg / ml peptide. For each peptide tested, threshold activity was determined. It is defined as the lowest concentration of the test peptide, which induced an intracellular level of cyclic GMP more than two standard deviations above the control. Control samples had intracellular cyclic GMP values of less than 0.9 pmol / ml (mean ± SD). Test results were considered positive if the level of cyclic GMP was more than 2 times the level that was determined for the parallel negative control sample. Active peptides had activity thresholds ranging from 1 to 10 g / L.

 The table illustrates the results of cyclic GMP analysis. Positive (+) results show that the level of action of cyclic GMP is more than 2 times higher than the level determined for parallel negative control.

 PRI me R 9. Enzyme resistance.

To illustrate the stability of a peptide to degradation by intestinal and serum enzymes, the peptide Arg-Pro-Asp-Val-isopropylamide can be incubated at ³⁷ ° C in intestinal juice and human serum for up to 120 minutes. For comparison, peptides such as Arg-Lys-Asp-Val-dimethylamide and thymopentin are similarly processed. Analysis by high performance liquid chromatography showed that Arg-Lys-Asp-Val-dimethylamide is fully digested after only a few seconds. Under these conditions, thymopentyl decomposes by 50% for about 20 s. The proposed peptides remain practically undecomposed both in serum and in intestinal juice for a longer time than thymopentin or a control peptide.

Claims (4)

1. Peptides of the general formula I
Arg Lys Asp Val NR ₁ R ₂ ,
where —NR ₁ R _{2 is} selected from the group consisting of anilinamide, methylamide, 2-phenylhydrazine, piperidinamide, cyclohexylamide and isopropylamide.  2. The peptides according to claim 1, characterized in that they have the ability to regulate insufficient or excessive T-cell function of the body. 3. A composition capable of regulating the immune function of mammals, comprising a peptide and a pharmaceutically acceptable carrier, characterized in that the peptide is represented by a compound of the general formula
Arg Lys Asp Val NR ₁ R ₂
where —NR ₁ R _{2 is} selected from the group consisting of anilinamide, methylamide, 2-phenylhydrazine, piperidinamide, cyclohexylamide and isopropylamide,
in an amount of 0.001 to 100 mg / kg of mass.  4. The composition according to claim 3, characterized in that it is suitable for oral administration.

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