RU2482128C1 - Peptides possessing cytoprotective activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to organic chemistry, more specifically to new biologically active peptides which can be used in pharmacology and medicine to create new drug preparations that have cytoprotective activity.

EFFECT: new biologically active peptides are presented.

12 ex, 28 dwg, 2 tbl

Description

The invention relates to organic chemistry, specifically to new biologically active peptides of the general formula:

A-L-Asp-L-Glu-B

where A-H is the residue of an amino acid or acyl;

B-OH is the residue of an amino acid or any organic compound capable of forming an amide or ester chemical bond with cytoprotective activity, which can be used in pharmacology and medicine to create new drugs.

A number of short peptides are known, containing in their structure derivatives of the Y-L-Glu-L-Asp-X dipeptide and having the properties of intercellular mediators [1-5]. The disadvantage of these compounds is their chemical instability and a tendency to spontaneous rearrangements.

If X is a glycine residue, spontaneous migration of this residue from α-carboxyl of aspartic acid to β-carboxyl occurs. And although this transformation can occur in the case of any amino acid, the rate of this reaction strongly depends on steric hindrances [6-7]. If X is a proline residue or any other organic compound with a secondary amino group, then a spontaneous breaking of the bond between these residues occurs [8].

It should be noted that these transformations lead to partial racemization of the aspartic acid residue.

The purpose of the claimed technical solution is to obtain chemically stable peptides with cytoprotective activity. The goal is achieved by the described peptides of the general formula:

A-L-Asp-L-Glu-B

where A- = H is the residue of an amino acid or acyl;

B- = OH is the residue of an amino acid or any organic compound capable of forming an amide or ester chemical bond.

The synthesis of the claimed compounds is carried out using generally accepted in chemistry biologically active peptides teaching methods [9-10] and can be easily scaled to industrial volumes.

The claimed compounds may find application in the food, cosmetic or pharmaceutical industries. For substances used in these areas, one of the main criteria for applicability is chemical storage stability, as well as stability in the composition of the final products. Accepted Conditional Abbreviations:

Boc - tert-butyloxycarbonyl

Bzl - benzyl

Z - benzyloxycarbonyl

Bu - tert-butyl

DCHA - Dicyclohexyl Amine

TFA - trifluoroacetic acid

ONP - para-nitrophenyl

ONSu - N-Oxysuccenyl

NMM - N-methylmorpholine

DMF - dimethylformamide

Tos - para-toluenesulfonate

DCHA - Dicyclohexylamine

Ala (A) - Alanine residue

Asp (D) - Aspartic Acid Residue

Glu (E) - Glutamic Acid Residue

Lys (K) - lysine residue

Gly (G) - glycine residue

Arg (R) - arginine residue

Trp (W) - tryptophan residue

Leu (L) - Leucine residue

DMEM - Dulbecco's Modified Eagle's Medium, Dulbecco's Modified Needle Medium

FBS - fetal bovine serum, cow fetal serum

PBS - phosphate buffer saline, phosphate buffered saline, pH 7.4

HeLa (ATCC Number - CCL-2,2, subclone S3) - human uterine adenocarcinoma cells epithelial morphology

NIH 3T3 (ATCC Number - CRL-1658) - Mouse Embryonic Fibroblasts

DMSO - dimethyl sulfoxide

EDTA Ethylene Diamine Tetra Acetate

The invention is illustrated by the following examples:

Example 1. H-Asp-Glu-OH (DE)

1. Boc-Asp (OBzl) -Glu (OBzl) -OBzl_

To a solution of 48.5 g (150 mmol) of Boc-Asp (Bzl) -OH in 300 ml of DMF cooled to -20 ° C, 16.5 ml (150 mmol) of NMM and 19.5 ml (150 mmol) of isobutyl chloroformate were added and stirred within 15 minutes The mixture was stirred at the same temperature for 20 minutes. Then a solution of 74.5 g (155 mmol) of the amino component H-Glu (Bzl) -OBzlTos in 200 ml of DMF containing 17 ml of NMM (155 mmol) was added cooled to -20 ° C. The reaction mixture was stirred for 1 hour at -10 ° C, for 2 hours at room temperature. Evaporated, the residue was dissolved in ethyl acetate (500 ml) and washed successively with 5% NaHCO ₃ , H ₂ O, 2% H ₂ SO ₄ , H ₂ O (twice 200 ml each), evaporated, evaporated again with isopropyl alcohol. 500 ml of hexane and 50 ml of ether were added to the resulting oil, and the resulting oil was triturated until solid particles formed. The product crystallizes in the refrigerator. The precipitate was filtered off, washed on the filter with hexane, and dried. Received 89 g (89%) of a chromatographically homogeneous product.

2. H-Asp-Glu-OH

12.64 g (20 mmol) of the protected Boc-Asp (Bzl) -Glu (Bzl) -OBzl dipeptide was dissolved in 100 ml of chloroform and 100 ml of trifluoroacetic acid was added to the resulting solution at 0 ° C, the mixture was kept for 1 hour at room temperature and the solvents were evaporated in vacuo. The residue was dissolved in 300 ml of ethyl acetate and washed with 5% bicarbonate solution 3 times in 200 ml. Ethyl acetate was evaporated, the residue was dissolved in 500 ml of 80% acetic acid and hydrogenated over 5% Pd / C at room temperature. After completion of hydrogenation (TLC control Rf system: chloroform: methanol: 32% acetic acid - 60:45:20), the catalyst was filtered off, the filtrate was evaporated, the residue was dissolved in 300 ml of water, re-evaporated to half volume and lyophilized. Received 4.8 g (91%) of the target dipeptide. According to HPLC, the purity of the product is more than 95%.

Example 2. H-Ala-Asp-Glu-OH (ADE)

1.Z-Ala-Asp (OBzl) -Glu (OBzl) ₂

19 g (30 mmol) of Boc-Asp (OBzl) -Glu (OBzl) _{2 was} dissolved in 200 ml of TFA, evaporated after 1 hour, the residue was dissolved in 500 ml of dry DMF and Na ₂ CO _{3 was} added in portions until the evolution of CO ₂ ceased. The precipitate was filtered off, washed with 100 ml of DMF, 3.33 ml (30 mmol) of N-methylmorpholine and 9.72 g (30 mmol) of Z-Ala-ONSu were added and left at room temperature for 15 hours. The reaction mixture was evaporated by 80%, 800 ml of isopropyl alcohol was added to the resulting syrupy residue, held for 2 hours at + 4 ° C, the fine precipitate was filtered off, washed with isopropyl alcohol, ether, and dried. Yield 20.3 g (92%) of product.

2. H-Ala-Asp-Glu-OH

20.3 g (28 mmol) of the protected tripeptide Z-Ala-Asp (OBzl) -Glu (OBzl) _2, when heated to + 50 ° C, was dissolved in 1000 ml of AcOH + 100 ml of water and hydrogenated over 5% Pd / C, periodically heating the reaction mixture. The progress of the reaction was monitored by TLC. After completion, the catalyst was filtered off, the filtrate was evaporated to a liquid oil consistency, and 800 ml of isopropyl alcohol was added. The precipitate formed was filtered off, washed with isopropyl alcohol, ether, and dried. The substance was dissolved in deionized water, the residual isopropyl alcohol was evaporated and lyophilized. Yield 7.67 g (85%) of product. HPLC more than 95%.

In the PMR spectrum:

Ala - 3.81 (α-CH); 1.35 (β-CH ₃ ); 8.08 (NH ₂ )

Asp - 4.62 (α-CH); 2.70; 2.53 (β-CH ₂ ); 8.63 (NH)

Glu - 4.20 (α-CH); 1.97; 1.78 (β-CH ₂ ); 2.26 (γ-CH ₂ ); 8.19 (NH).

Example 3. H-Lys-Asp-Glu-OH (KDE)

1.Z-Lys (Z) -Asp (OBzl) -Glu (OBzl) ₂

19 g (30 mmol) of Boc-Asp (OBzl) -Glu (OBzl) ₂ (1) was dissolved in 200 ml of TFA, evaporated after an hour, the residue was dissolved in 500 ml of dry DMF and Na ₂ CO _{3 was} added in portions until the evolution of CO ₂ ceased . The precipitate was filtered off, washed with 100 ml of DMF, 3.33 ml (30 mmol) of N-methylmorpholine and 15.36 g (30 mmol) of Z-Lys (Z) -ONSu were added and left at room temperature for 15 hours. The reaction mixture was evaporated, the resulting syrupy residue was dissolved in 300 ml of ethyl acetate and washed successively with 2% sulfuric acid, water until neutral, 5% soda solution and again with water. Ethyl acetate was evaporated and 500 ml of isopropyl alcohol was added to the oily residue, held for 2 hours at + 4 ° C, the precipitate was filtered off, washed with isopropyl alcohol, ether, and dried. Yield 21.6 g (90%) of product.

2. H-Lys-Asp-Glu-OH

21.6 g (27 mmol) of the compound Z-Lys (Z) -Asp (OBzl) -Glu (OBzl) ₂ , prepared as described above, was dissolved by heating in 0.5 L of glacial acetic acid, 50 ml of water was added and hydrogenated in the presence of 5 g of a palladium catalyst. The catalyst was filtered off and the solvents were evaporated. Crystallized from a mixture of acetic acid-ethanol. If necessary, the product is purified by ion-exchange chromatography on a column with SP-Sephadex, applied in water, washed with 0.04 M pyridine-acetate buffer (pH 5.4). Fractions containing the desired compound were evaporated. The syrupy residue was dissolved in 300 ml of water, evaporated to half the volume, treated with activated carbon and lyophilized. Yield 8.95 g (85%) of product. The purity of the product according to HPLC more than 95%.

In the PMR spectrum:

Lys 3.76 (α-CH); 1.72 (β-CH ₂ ); 1.36; 1.52 (γ-CH ₂ ); 2.74 (ε-CH ₂ ); 8.11 (NH ₂ )

Asp - 4.63 (α-CH); 2.71; 2.54 (β-CH ₂ ); 8.68 (NH)

Glu - 4.19 (α-CH); 1.98; 1.78 (β-CH ₂ ); 2.28 (γ-CH ₂ ); 8.31 (NH).

Example 4. H-Asp-Glu-Pro-OH (DEP)

1. Z-Asp (OBu ^t ) -Glu (OBu ^t ) OH DCHA

17.5 g (100 mmol) of H-Glu (OBu ^t ) -OH was dissolved (suspended) in methanol, 50 ml of 40% Triton B in methanol was added and, after complete dissolution, evaporated in vacuo. The residue was dissolved in 300 ml of dimethylformamide, evaporated by a quarter and 42.0 g (100 mmol) of Z-Asp (OBu ^t ) -ONSu was added. After 12 hours (control - TLC in the system chloroform: methanol: acetic acid - 9: 1: 0.5 “B”), the reaction mixture was evaporated, the residue was dissolved in ethyl acetate, washed successively with 2% sulfuric acid, water, dried over Na ₂ SO ₄ , evaporated. The residue was dissolved in 150 ml of ether, 20 ml (100 mmol) of dicyclohexylamine was added and left at room temperature until a precipitate formed completely. The precipitate was filtered off, washed with ether, and dried in air. Yield 60 g (87%) of a chromatographically homogeneous substance.

2. H-Asp-Glu-Pro-OH

28 g (40 mmol) of Z-Asp (OBu ^t ) -Glu (OBu ^t ) -OH · DCHA were suspended in 500 ml of ethyl acetate, washed with 2% sulfuric acid, water, dried over Na ₂ SO ₄ , the drying agent was filtered off, the filtrate was evaporated, the residue was dissolved in 150 ml of dimethylformamide, cooled to -25 ° C, 4.4 ml (40 mmol) of N-methylmorpholine and 5.2 ml (40 mmol) of isobutyl chloroformate were added to the cooled solution, kept at -20 ° C for 15 min and a solution of 5.6 g (44 mmol) of N-hydroxysuccinimide 25 ° C in 50 ml of dimethylformamide cooled to -25 ° C was added. After 30 minutes, a solution of 10.6 g (44 mmol) of H-Pro-OBzl HCl and 5 ml of N-methylmorpholine in 50 ml of dimethylformamide was added and left at room temperature overnight. The reaction mixture was evaporated, the residue was dissolved in ethyl acetate and washed in the usual way. Ethyl acetate was evaporated, the residue was evaporated with toluene. The resulting oil was dissolved in TFA, kept for an hour, evaporated, the residue was treated with an ether: hexane 1: 1 mixture, the precipitate was filtered off, washed on the filter with an ether - hexane 1: 1 mixture, hexane, and dried over an alkali in a desiccator.

The resulting product was dissolved in ethanol - water - acetic acid (600: 100: 100) and hydrogenated over 5% Pd \ C. The catalyst was filtered off and the filtrate was evaporated. The residue was reprecipitated from acetic acid with isopropyl alcohol, filtered, washed on the filter with isopropyl alcohol, ether, and dried. Yield 12.2 g (85%). Purity according to HPLC more than 95%.

In the PMR spectrum:

Asp - 4.13 (α-CH); 2.79; 2.66 (β-CH ₂ ); 8.14 (NH ₂ )

Glu - 4.59 (α-CH); 1.93; 1.72 (β-CH ₂ ); 2.36 (γ-CH ₂ ); 8.67 (NH)

Pro - 4.23 (α-CH); 2.14; 1.84 (β-CH ₂ ); 1.90 (γ-CH ₂ ); 3.63 (δ-CH ₂ ).

Example 5. H-Asp-Glu-Arg-OH (DER)

1. Z-Glu (OtBu) -ArgOH

To a solution of 104.4 g (228 mmol) of Z-Glu (OtBu) ONP in 0.5 L of dimethylformamide was added 38.3 g (228 mmol) of H-Arg-OH. The reaction mixture was stirred at room temperature for 24 hours. Then it was kept at -4 ° C for four hours, the precipitate formed was filtered off and washed on a filter with 0.4 L of a mixture of dimethylformamide-ether (1: 2), 0.45 L of ether, 0.4 L of hexane. Dried. Yield: 91.7 g (82.3%) of chromatographically homogeneous Z-Glu (OtBu) ArgOH (control in the system: chloroform-methanol-acetic acid (5: 3: 1)).

2. H-Asp-Glu-Arg-OH

A solution of 90.6 g (185 mmol) of ZGlu (OtBut) ArgOH in 1.2 L of ethanol was hydrogenated over 10 g of 5% Pd / C. After hydrogenation (TLC control in the system: chloroform-methanol-acetic acid (5: 3: 1)), the catalyst was filtered off, the solvent was evaporated. The resulting syrupy product was dissolved in 500 ml of dimethylformamide, and 75.9 g (185 mmol) of Boc-Asp (OtBu) -ONP was added to the resulting solution. The reaction mixture was left at room temperature for 15 hours until completion of the reaction (control in the system: chloroform-methanol-acetic acid (5: 3: 1)). The solvent was evaporated, the resulting oil was dissolved in 150 ml of chloroform and applied to a column of silica gel (400 g), washed with chloroform until nitrophenol disappeared, 10% ethyl alcohol in chloroform, washed with 30% ethyl alcohol in chloroform, and evaporated (control in the system: chloroform- methanol-acetic acid (5: 3: 1)). 400 ml of trifluoroacetic acid was added to the residue, and the mixture was kept for 2 hours at room temperature. The acid was evaporated, 1000 ml of diethyl ether was added to the residue, the amorphous precipitate formed was filtered off, washed with ether twice in 150 ml filter and dried. The substance was dissolved in 1500 ml of water, evaporated to an oil, re-dissolved in 1000 ml of water and applied to an SP-Sephadex column. The column was washed with two volumes of 0.05 M pyridine acetate buffer and the material was washed with the same 0.2 M buffer. The product containing fractions were combined, the solvents were evaporated, re-evaporated with water to remove pyridine acetate residues and lyophilized. Received 65 g (75%) of product with a purity according to HPLC more than 95%.

In the PMR spectrum:

Asp - 4.16 (α-CH); 2.82; 2.65 (β-CH ₂ ); 8.14 (NH ₂ )

Glu - 4.35 (α-CH) 1.93; 1.87 (β-CH ₂ ); 2.30 (γ-CH ₂ ); 8.60 (NH)

Arg - 4.16 (α-CH); 1.75; 1.62 (β-CH ₂ ); 1.54; 1.50 (γ-CH ₂ ); 3.10 (δ-CH ₂ ); 7.58 (ε-NH ₂ ); 8.28 (NH).

Example 6. H-Asp-Glu-Gly-OH (DEG)

1. Boc-Glu (BzI) -Gly-Bzl

To a solution of 8.43 g (25 mmol) of Boc-Glu (Bzl) -OH in 100 ml of DMF cooled to -20 ° C, 2.77 ml (25 mmol) of NMM and 3.25 ml (25 mmol) of isobutyl chloroformate were added, stirred a solution of 7.97 g (25 mmol) of the amino component H-Gly-OBzl × Tos in 50 ml of DMF containing 3 ml of NMM (27 mmol) cooled to -20 ° C was added. The reaction mixture was stirred for 1 hour at -10 ° C, for 2 hours at room temperature. Evaporated, the residue was dissolved in ethyl acetate (200 ml) and washed successively with 5% NaHCO ₃ , H ₂ O, 2% H ₂ SO ₄ , H ₂ O (twice 200 ml each), evaporated, evaporated again with isopropyl alcohol. The product crystallizes from a mixture of isopropyl alcohol-hexane 1: 1 at + 4 ° C in the refrigerator. The precipitate was filtered off, washed on the filter with hexane, and dried. Received 11 g (91%) of a chromatographically homogeneous product.

2. Z-Asp (Bzl) -Glu (Bzl) -Gly-Bzl

10 g (20.7 mmol) of Boc-Glu (OBzl) -Gly-OBzl was dissolved in 50 ml of TFA, evaporated after an hour, the residue was dissolved in 100 ml of dry DMF and Na ₂ CO _{3 was} added in portions until the evolution of CO ₂ ceased. The precipitate was filtered off, washed with 50 ml of DMF, 9.53 g (21 mmol) of Z-Asp (Bzl) -ONSu was added to the resulting solution, and left at room temperature for 15 hours. The reaction mixture was evaporated, the resulting syrupy residue was dissolved in 300 ml of ethyl acetate and washed successively with 2% sulfuric acid, water until neutral, 5% soda solution and again with water. Ethyl acetate was evaporated and 100 ml of isopropyl alcohol was added to the oily residue, held for 2 hours at + 4 ° C, the precipitate was filtered off, washed with isopropyl alcohol, ether, and dried. Yield 12 g (80%) of a chromatographically homogeneous product.

3. H-Asp-Glu-Gly-OH

7.23 g (27 mmol) of Z-Asp (Bzl) -Glu (Bzl) -Gly-Bzl, prepared as described above, was dissolved by heating in 0.5 L of glacial acetic acid, 50 ml of water was added and hydrogenated in the presence of 5 g of palladium catalyst. The catalyst was filtered off and the solvents were evaporated. The product was reprecipitated from acetic acid-isopropyl alcohol mixture. Received 3.0 g (94%). The purity of the product according to HPLC more than 95%.

In the PMR spectrum:

Asp - 4.14 (α-CH); 2.87; 2.68 (β-CH ₂ ); 8.16 (NH ₂ )

Glu - 4.35 (α-CH) 1.94; 1.80 (β-CH ₂ ); 2.31 (γ-CH ₂ ); 8.63 (NH)

Gly 3.75 (α-CH ₂ ); 8.28 (NH).

Example 7. H-Ala-Asp-Glu-Leu-OH (ADEL)

1. Boc-Glu (Bzl) -Leu-OBzl

To a solution of 26.6 g (78 mmol) of Boc-Glu (Bzl) -OH in 200 ml of dimethylformamide and 8.65 ml of N-methylmorpholine, cooled to -15 ° C, 10.4 ml (80 mmol) of isobutyl chloroformate was added, mixture stirred at this temperature for 20 minutes and a solution of 30.5 g (78 mmol) of H-Leu-OBzl * Tos cooled to -15 ° C was added, the pH of which was previously adjusted to 8.5-9.0. After an hour, the solvents were evaporated, the residue was dissolved in 500 ml of ethyl acetate and washed successively with 400 ml of 2% sulfuric acid solution, water, 5% soda solution and water. The organic layer was separated and ethyl acetate was evaporated. The residue was crystallized from ether-hexane (1: 1). Received 37.8 g (90%) of a chromatographically homogeneous substance.

2. Boc-Asp (Bzl) -Glu (Bzl) -Leu-OBzl

27 g (50 mmol) of Boc-Glu (Bzl) -Leu-OBzl was dissolved in 100 ml of chloroform, and 100 ml of trifluoroacetic acid was added to the resulting solution. The mixture was kept at room temperature for 2 hours and the solvents were evaporated. The residue was dissolved in 200 ml of dimethylformamide and soda was added in portions until the end of CO ₂ evolution. The precipitate was filtered off, washed with 50 ml of dimethylformamide, the pH was adjusted to 8.5 in the filtrate, and cooled to -20 ° C (Solution 1).

At the same time, 5.6 ml of N-methylmorpholine was added to a solution of 16.5 g (50 mmol) of Boc-Asp (Bzl) -OH in 100 ml of dimethylformamide, the mixture was cooled to -20 ° C and 6.5 ml (50 mmol) of isobutyl chloroformate was added . The mixture was kept at this temperature for 30 minutes (Solution 2).

While cooling, both solutions were mixed and allowed to mix until room temperature was reached (approximately 2 hours). The solvents were evaporated, the residue was dissolved in 300 ml of ethyl acetate and washed successively with 2% sulfuric acid solution, water, 5% soda solution, water and the solvent was evaporated. After crystallization from ether, 32.4 g (87%) of chromatographically homogeneous Boc-Asp (Bzl) -Glu (Bzl) -Leu-OBzl was obtained.

3.Z-Ala-Asp (Bzl) -Glu (Bzl) -Leu-OBzl

22.2 g (30 mmol) of Boc-Asp (Bzl) -Glu (Bzl) -Leu-OBzl was dissolved in 100 ml of chloroform, and 100 ml of trifluoroacetic acid was added to the resulting solution. The mixture was kept at room temperature for 2 hours and the solvents were evaporated. The residue was dissolved in 200 ml of dimethylformamide and soda was added in portions until the end of CO ₂ evolution. The precipitate was filtered off, washed with 50 ml of dimethylformamide, the pH was adjusted to 8.5 in the filtrate, and 9.6 g (30 mmol) of Z-Ala-ONSu was added. The reaction mixture was left for 15 hours at room temperature. The solvents were evaporated, the residue was dissolved in 500 ml of ethyl acetate and washed successively with 2% sulfuric acid solution, water, 5% soda solution, water and the solvent was evaporated. After crystallization from isopropyl alcohol, 22.4 g (87.8%) of chromatographically homogeneous Z-Ala-Asp (Bzl) -Glu (Bzl) -Leu-OBzl were obtained.

4. H-Ala-Asp-Glu-Leu-OH

17 g (20 mmol) of the protected tetrapeptide Z-Ala-Asp (Bzl) -Glu (Bzl) -Leu-OBzl was dissolved in 300 ml of 80% acetic acid and hydrogenated over 5% Pd \ C at room temperature. After completion of the reaction, the catalyst was filtered off, washed on the filter with acetic acid, and the solvents were evaporated. The residue was dissolved in hot water, cooled to room temperature and left at + 4 ° C for 15 hours. The precipitate was filtered off, dried in vacuo over KOH. Received 8 g (89%) of product with a purity according to HPLC more than 95%.

In the PMR spectrum:

Ala 3.83 (α-CH); 1.34 (β-CH ₃ ); 8.08 (NH ₂ )

Asp 4.61 (α-CH); 2.72; 2.54 (β-CH ₂ ); 8.68 (NH)

Glu - 4.28 (α-CH); 1.91; 1.76 (β-CH ₂ ); 2.23 (γ-CH ₂ ); 7.93 (NH)

Leu 4.17 (α-CH); 1.52 (β-CH ₂ ); 1.74; 1.62 (γ-CH ₂ ); 0.83 (δ-CH ₃ ); 0.89 (δ-CH ₃ ); 8.12 (NH).

Example 8. H-Asp-Glu-Leu-OH (DEL)

14.9 g (20 mmol) of the protected tripeptide Boc-Asp (Bzl) -Glu (Bzl) -Leu (Bzl) was dissolved in 50 ml of chloroform, and 50 ml of trifluoroacetic acid was added to the resulting solution. The mixture was kept for 1 hour at room temperature and the solvents were evaporated. The residue was dissolved in 300 ml of 80% acetic acid and hydrogenated over 5% Pd \ C at room temperature. After completion of the reaction, the catalyst was filtered off, washed on the filter with acetic acid, and the solvents were evaporated. 200 ml of isopropyl alcohol was added to the residue. The precipitate was filtered off, dried in vacuo over KOH. Received 7 g (93%) of product with a purity according to HPLC more than 95%.

In the PMR spectrum:

Asp - 4.13 (α-CH); 2.81; 2.65 (β-CH ₂ ); 8.15 (NH ₂ )

Glu - 4.34 (α-CH); 1.94; 1.80 (β-CH ₂ ); 2.30 (γ-CH ₂ ); 8.58 (NH)

Leu - 4.19 (α-CH); 1.52 (β-CH ₂ ); 1.53; 1.62 (γ-CH ₂ ); 0.83 (δ-CH ₃ ); 0.89 (δ-CH ₃ ); 8.22 (NH).

Example 9. H-Lys-Asp-Glu-Trp-NH ₂ (KDEW-NH2)

1. Z-Glu (OtBu) -Trp-NH ₂

To a solution of 35.4 g (100 mmol) of Z-Glu (OtBu) -OH in 200 ml of dimethylformamide and 11.1 ml of N-methylmorpholine, cooled to -15 ° C, 13 ml (100 mmol) of isobutyl chloroformate was added, the mixture was stirred at at this temperature for 20 minutes and a solution of 20.4 g (100 mmol) of H-Trp-NH ₂ cooled to -15 ° C was added, the pH of which was previously adjusted to 8.5-9.0. After an hour, the solvents were evaporated, the residue was dissolved in 500 ml of ethyl acetate and washed successively with 400 ml of 2% sulfuric acid solution, water, 5% soda solution and water. The organic layer was separated and ethyl acetate was evaporated. The residue was crystallized from ether-hexane (1: 1). 45.2 g (86%) of chromatographically homogeneous Z-Glu (OtBu) -Trp-NH _{2 were} obtained.

2. Z-Asp (OtBu) -Glu (OtBu) -Trp-NH ₂

To a solution of 24.7 g (76.5 mmol) of Z-Asp (OtBu) -OH in 300 ml of DMF cooled to -20 ° C, 8.5 ml (76.5 mmol) of NMM and 9.95 ml (765 mmol) were added. ) iBuOCOCl and mixed for 15 minutes. The mixture was stirred at the same temperature for 20 minutes. Then, a solution obtained by hydrogenation of 40 g (76.5 mmol) of Z-Glu (OtBu) -Trp-NH ₂ in 200 ml of DMF was added. The reaction mixture was stirred for 1 hour at -10 ° C, for 2 hours at room temperature. The completeness of the reaction was monitored in the chloroform: methanol: acetic acid system - 9: 1: 0.5. The solvent was evaporated, the residue was dissolved in ethyl acetate (500 ml) and washed successively with 5% NaHCO ₃ , H ₂ O, 2% H ₂ SO ₄ , H ₂ O (twice 200 ml each), evaporated, and again evaporated with isopropyl alcohol. The product crystallized from ethyl acetate-hexane (2: 1) at + 4 ° C in a refrigerator. The precipitate was filtered off, washed on the filter with hexane, and dried. 46.1 g (87%) of a chromatographically homogeneous product were obtained.

3. Z-Lys (Z) -Asp (OtBu) -Glu (OtBu) -Trp-NH ₂

40 g (57.6 mmol) of the protected tripeptide obtained as described above

Z-Asp (OtBu) -Glu (OtBu) -Trp-NH _{2 was} dissolved in 400 ml of alcohol and hydrogenated over 5% Pd \ C. After completion of the reaction, the catalyst was filtered off, the alcohol was evaporated, the residue was dissolved in 200 ml of dimethylformamide, and 29.5 g (57.6 mmol) of Z-Lys (Z) -ONSu were added to the resulting solution. The mixture was left for 15 hours at room temperature. The solvent was evaporated, the residue was dissolved in hot ethyl acetate and allowed to crystallize. The precipitate formed was filtered off, washed with ethyl acetate-hexane (1: 1), and dried. Received 45 g (82%) of a chromatographically homogeneous substance.

4. H-Lys-Asp-Glu-Trp-NH ₂

40 g (41.8 mmol) of Z-Lys (Z) -Asp (OtBu) -Glu (OtBu) -Trp-NH _{2 was} dissolved in 500 ml of alcohol, 5 g of 5% Pd \ C in 50 ml of water was added and hydrogenated to the end of the reaction. The catalyst was filtered off and the solvents were evaporated. The residue was treated with 200 ml of trifluoroacetic acid containing 1% mercaptoethanol. After 1 hour, the acid was evaporated and 500 ml of ether was added to the residue. The precipitate was filtered off, washed on the filter with ether and dried. It was dissolved in 100 ml of water and applied to a column with SP-Sephadex (or a similar cation exchanger) equilibrated with 0.05 M pyridine-acetate buffer. Washed with two volumes of start buffer, one volume of 0.1 M buffer and the substance was washed with 0.2 M buffer. The fractions containing the target product were combined, the solvents were evaporated, re-evaporated with 300 ml of water to remove traces of the pyridine-acetate buffer. The residue was dissolved in 400 ml of water and lyophilized. Received 26 g (79%) of a chromatographically homogeneous product with a purity according to HPLC more than 95%.

In the PMR spectrum:

Lys - 3.75 (α-CH), 1.70 (β-CH ₂ ), 1.51; 1.34 (γ-CH ₂ ), 2.73 (ε-CH ₂ ), 8.12 (NH ₂ )

Asp = 4.61 (α-CH), 2.55; 2.72 (β-CH ₂ ), 8.73 (NH)

Glu - 4.19 (α-CH), 1.86; 1.73 (β-CH ₂ ), 2.18 (γ-CH ₂ ), 8.16 (NH)

Trp - 4.43 (α-CH), 3.14, 2.98 (β-CH ₂ ), 7.84 (NH); 7.58; 6.96; 7.05; 7.81 (CH aromatic); 10.8 (NH indole).

Example 10. The study of the stability of the peptides during storage.

Peptides were dissolved in 0.05 M ammonium acetate buffer with a pH of 4.5 and a pH of 7.5 so that their concentration was about 1 mg / ml. The solutions were sterile filtered and stored in sterile, well-sealed vials. After the expiration date, the substances were analyzed by HPLC. The results are presented in table 1.

Table 1 Peptide stability during storage Peptide Duration (time, day) 1 hour 10 thirty 60 90 Storage conditions (pH, 22 ° C) 4.5 7.5 4.5 7.5 4.5 7.5 4.5 7.5 4.5 7.5 ADE 97.2 97.2 97.15 97.2 97.1 97.13 97.1 97.05 97.15 96.9 DER 98.1 98.1 98.1 98.1 97.9 97.7 98.05 97.6 97.9 97.5 DEP 96.5 96.5 96.5 96.45 96.4 96.3 96.5 96.2 96.3 95.7 Deg 96.7 96.7 96.7 96.7 96.6 96.5 96.3 96.2 96.2 95.8 ADEL 98.2 98.2 98.3 98.2 98.1 98.05 98.1 98.1 98.0 97.8

The results are presented as the purity of the investigated products, expressed as a percentage.

The presented analysis results indicate that the products are well stored even in solutions at various pH values.

Example 11. The study of the biological activity of peptide preparations under standard cell culture

The biological activity of the synthesized peptides was studied in five different concentrations (400, 200, 100, 10, and 1 ng / ml) using cultured mouse and human cells: HeLa S3 and NIH 3T3 as a model system.

The biological activity of each peptide was determined by the following parameters: the effect of peptides on the proliferative activity of cells and the ability of peptides to cause apoptosis and necrosis. For this, the following indicators were investigated: the percentage of dead cells, the total number of cells in the vial, the level of apoptotic cell death.

Cell cultivation and peptide treatment

Cultivation of NIH ZTZ fibroblasts was performed in complete nutrient medium DMEM (Paneko, RF) containing 10% fetal serum (Paneko), gentamicin 2 u / ml (Paneko), glutamine 29.2 μg / ml (Paneko), in a CO ₂ incubator . For cultivation used bottles of the company SPL (South Korea) with an area of 25 cm ² and 175 cm ² . Reseeding of cells was carried out in a ratio of 1: 5-1: 7. Upon reaching approximately 50% confluency, peptides were added to the vials at the indicated final concentrations. In each experiment, two vials were used with the same concentration of each peptide. An appropriate volume of complete medium without peptides was added to control vials. Two independent experiments were carried out with each peptide (two replicates in each). After 24 hours incubation with peptides, the cells were removed from the culture vial by treatment in a mixture of trypsin and versene (1: 3, Paneko). Cells from each vial were placed in the same volume of buffered saline (0.01 M PBS, pH 7.2). The cell suspension was divided into 3 equal parts to study the above indicators.

The cultivation of HeLa cells was carried out in a similar manner, except that cell reseeding was carried out in a ratio of 1: 3-1: 4.

The study of indicators was carried out using flow cytometry. Used a flow cytofluorimeter sorter FACS Vantage (Becton Dickinson, USA). Before each series of analyzes, the instrument was aligned and calibrated using standard Microspheres (Polyscience) in accordance with the methodology recommended by the manufacturer.

Determination of the total number of cells in the vial and the proportion of dead cells

Propidium iodide (prodium iodide-PI, Sigma, USA) at a final concentration of 10 μg / ml and Calibrite calibration particles (Becton Dickinson, USA) labeled with fluorescein isothiocyanate (FITCem) were added to the cell suspension obtained as described above. The final concentration of calibration particles was determined using a Goryaev camera. They were incubated for 10-12 minutes and immediately analyzed on a flow cytometer using 4 parameters: direct light scattering, lateral light scattering, PI fluorescence, FITC fluorescence. In each sample, 20 thousand cells were analyzed. Data was written to a file. Then, computer processing of data was performed using the CellQuestPro program (Becton Dickinson, USA).

The proportion of PI + cells with a damaged plasma membrane (dead cells) was determined. The death rate in control bottles (15-20 pieces) was averaged and taken as 100%. The possible effect of peptides was evaluated by the ratio with the control.

FITC + calibration particles were detected in each file and their number was determined. Then, cells were identified by light scattering and their number was found in the same files. The ratio of cells / calibration particles in each file (sample) was calculated, which was used to judge the number of cells in the vial. The ratio of cells / calibration particles in the control was taken as 100%. The number of cells in the experimental vials was evaluated as a percentage compared with the control.

Determination of cell death by apoptosis

The method is based on the fact that fragmented DNA appears in cells undergoing apoptotic death, which is removed from the cells during treatment with HCl solution. Then, the DNA remaining in the cells is stained with specific dyes (for example, PI) and cell-by-cell DNA content is determined. Apoptotic cell death is assessed by the proportion of cells with a hypodiploid DNA content.

The cell suspension obtained as described above was cooled to + 4 ° C. Chilled ethanol was added to the cell suspension in a volume ratio of 1: 3 with constant stirring. Cells were stored 1-2 weeks prior to analysis. By centrifugation, ethanol was removed from the samples, then the fragmented DNA was removed from the cells with 0.6 M HCl (37 ° C, 10 min). Then the cells were washed with PBS and stained in 0.3 ml of a PI solution (50 μg / ml) in PBS, kept for 15 min at room temperature and analyzed on a flow cytometer. PI at this concentration allows for the determination of cell-by-cell DNA content and to identify the fraction of cells with fragmented DNA with high resolution. To measure PI fluorescence, narrow-band filters of 585/42 nm were used; the laser power was 57 mW. In each sample, 5 × 10 ³ cells were analyzed, then computer processing was performed using the CellQuestPro program (Becton Dickinson, USA). At the first stage of the analysis, a region of cells was isolated by light scattering, then PI fluorescence was evaluated in this region. A marker of hypodiploid DNA content was set in control samples. The results are shown in Fig. 1 - Fig. 12.

Example 12. The study of the protective properties of peptides

In experiments on the effect of peptides on apoptosis and necrosis, the ApoTarget Annexin-V FITC Apoptosis Kit reagent kit (Invitrogen, United States) was used.

Apoptosis is a programmed cell death resulting from the implementation of a genetic program or a response to external factors and requiring energy and synthesis of macromolecules. Apoptosis is accompanied by the appearance of characteristic cytological features (markers of apoptosis) and molecular processes. In living cells, in particular, an asymmetric distribution of various phospholipids between the inner and outer monolayers of the cytoplasmic membrane is observed: phospholipids containing choline, such as phosphatidylcholine and sphingomyelin, are located mainly in the outer monolayer, and phosphatidylethanolamine and phosphatidylsulfinamine (amino). During apoptosis, the cytoplasmic membrane undergoes changes, one of which is the transition of phosphatidylserine from its inner monolayer to the outer one, where phosphatidylserine is available for binding to Annexin-V labeled with FITC fluorescent dye. Annexin-V - calcium-dependent, phospholipid-binding protein with mol. weighing 35-36 kDa, having a high affinity for phosphatidylcholine (K _d equal to 5 × 10 ^-10 M). Annexin-V-binding assay is based on the rapid and highly specific identification of cells containing phosphatidylcholine in the outer monolayer of the cytoplasmic membrane, i.e. cells at an early stage of apoptosis.

In addition to Annexin-V labeled with FITC fluorescent dye, the ApoTarget Annexin-V FITC Apoptosis Kit reagent kit contains another dye - propidium iodide (PI). PI has the ability to penetrate only those cells whose integrity of the cytoplasmic membrane is impaired, which is one of the characteristic signs of the late stages of apoptosis or necrosis. Penetrating cells with a damaged cell membrane, PI binds to DNA. The "red" fluorescence of the resulting complexes is detected using a flow cytometer.

Thus, using the double cell staining procedure (Annexin-V FITC plus PI) and flow cytometry, we can distinguish 3 cell populations and determine their percentage: 1) non-apoptotic cells (Annexin-V-negative and PI-negative), 2) cells that are at an early stage of apoptosis (Annexin-V-positive and PI-negative), 3) necrotic cells or cells that are at a late stage of apoptosis (Annexin-V-positive and PI-positive). By increasing the proportion of apoptotic and necrotic cells, one can judge the degree of influence of compounds added to the culture medium on the mentioned processes. The camptothecin compound (Camptothecin, Sigma, USA) recommended by the manufacturer, which has a pronounced ability to cause apoptosis and necrosis of cells, especially tumor cells, was used as a positive control in the experiments.

Solutions of peptides in sterile PBS with concentrations of 1 mg / ml were prepared immediately before experiments to study the effect of peptides on apoptosis and necrosis. To study proliferative activity, the same solutions were used that, after preparation, were stored in sterile tubes at + 4 ° C.

On the day of each experiment, working dilutions of peptides in cell culture medium were prepared. A sterile plastic 96-well plate (Corning, USA) was used for this. Dilutions were prepared so that when 25 μl of each working dilution was added to the experimental well with cultured cells, the final peptide concentrations were 400, 200, 100 and 50 ng / ml.

Cultured cells

Invitrogen reagents (USA) and plastic bottles, tablets, pipettes and filters from Corning (USA) were used to cultivate HeLa S3 and NIH ZTZ cells.

Cells were cultured in DMEM prepared from a dry concentrate dissolved in pyrogen-free distilled water (Millipore, USA). The medium contained 10% FBS, 2 mM glutamine and penicillin-streptomycin (diluted in accordance with the manufacturer's recommendations). HeLa S3 and NIH 3T3 were grown in T150 bottles at + 37 ° C in an atmosphere of 5.6% CO ₂ . Cells were constantly maintained in the logarithmic growth phase, for which they were scattered at least 2 times a week, using trypsin to remove attached cells from the surface of the culture vials. Live cells were counted in a hemacytometer after staining an aliquot of the suspension with trypan blue.

Study of the ability of peptides to induce apoptosis and necrosis of cultured cells

HeLa S3 and NIH 3T3 cells were removed from the surface of the culture vials, counted, the concentration was adjusted to 10 ⁵ cells / ml and the resulting suspensions were dispersed 1 ml into the wells of 24-well culture plates.

The next day, making sure that the cells attached to the surface of the plates, the culture medium in all wells was changed to fresh.

Camptothecin was added to control wells (positive control — stimulation of apoptosis and necrosis) to a final concentration of 50 μM, which was determined in preliminary experiments with NIH 3T3 and HeLa S3 cells. 25 μl of fresh culture medium was added to control wells (negative control), 25 μl of working dilutions of peptides (each working dilution in 2 repetitions) and camptothecin in the same final concentration were added to experimental wells. Incubation was carried out at + 37 ° C.

24 hours after the start of incubation, cells were removed from the surface of 24-well plates, transferred from each well to a separate 1.5 ml centrifuge tube, washed with 1 ml of Annexin-V FITC dye binding buffer solution, and cells were pelleted by centrifugation (Sysmex Platelet Centrifuge RS-810). The supernatant was discarded, 5 μl of Annexin-V FITC dye and 10 μl of propidium iodide (PI) dye were added to the cell pellet and incubated for 15 min in the dark at room temperature (+ 25 ° C). Then, 300 μl of Annexin-V FITC binding buffer solution was added to each tube and the green and red fluorescence intensities were measured.

Two populations were identified in the obtained distributions: Annexin-V-positive and PI-negative (cells undergoing apoptosis) and Annexin-V-positive and PI-positive cells (cells undergoing necrosis).

The histograms Fig. 13 - Fig. 28 show the percentage of apoptotic and necrotic cells in the populations HeLa S3 and NIH3T3 in the presence of 8 different peptides in 4 different concentrations.

The histograms (Fig. 13-28) and Table 2 show that the studied peptides in the concentration range from 50 to 400 ng / ml have a pronounced protective effect on HeLa S3 and NIH3T3 cells, i.e. possess cytoprotective activity.

Literature

1. RF patent No. 2177802 (2002).

2. RF patent No. 2161501 (2001).

3. RF patent No. 2301074 (2007).

4 RF Patent No. 2304444 (2007).

5. RF patent No. 2155063 (2000).

6. Pharmactutical Research, Vol. 11, No. 5, 1994, p. 751-758.

7. Pharmactutical Research, Vol.7, No.8, 1990, p. 787-793.

8. Synthetic peptides, sec. ed. Gregory A. Grant, Oxford Univ. Press 2002, p. 283.

9. A.A. Gershkovich, V.K. Kibirev, Chemical synthesis of peptides. Kiev, Naukova Dumka, 1992

10. Peptides, the main methods of formation of peptide bonds. Moscow, Mir 1983

Claims (1)

A drug having cytoprotective activity, which is a peptide selected from the group: H-Ala-Asp-Glu-OH, H-Lys-Asp-Glu-OH, H-Asp-Glu-Pro-OH, H-Asp-Glu -Arg-OH, H-Asp-Glu-Gly-OH, H-Ala-Asp-Glu-Leu-OH, H-Asp-Glu-Leu-OH, H-Lys-Asp-Glu-Trp-NH ₂ .

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