RU2110275C1 - Method of synthesis of peptide showing anabolic activity, stimulating body mass increase, development of epidermal layer and hair growth - Google Patents

Abstract

FIELD: medicine, pharmacology. SUBSTANCE: method of synthesis involves the successive chain splicing from C-end of molecule using strategy of maximal blocking functional groups. Pentapeptide is synthesized from glycine tert. -butyl ester using a method of activated esters and a method of mixed anhydrides and using carbobenzoxy- and tert. -butylhydroxycarbonylamino acids. Final deblocking is carried out in acid medium and the end product is purified by ion-exchange chromatography. Peptide can be used in clinics, veterinary science and experimental investigations. EFFECT: improved method of synthesis. 8 dwg

Description

 The invention relates to medicine, namely to pharmacology, more specifically to methods for producing biologically active substances, and can find application in the clinic, veterinary medicine, as well as in experimental studies.

 The relevance of developing methods for producing new biologically active substances that regulate anabolic processes leading to an increase in body weight in animals and an increase in the growth and quality of hairline is confirmed by a large number of studies conducted in this area.

 As a rule, the solution to this problem comes down either to the creation of whole compositions, including vitamins, hormones and other biological active substances (SU N 1664325, 1988; GB N 1460020, 1976), or to the search for optimal technologies for the purification of natural growth hormone that affects the process hair growth and body weight (US N 4115375, 1978; US N 4332717, 1982; US N 4371462, 1983), or obtained by synthesis of agents similar to growth hormone (EP N 0137904, 1984). In this case, a special group consists of synthesized physiologically active substances of a peptide or polypeptide nature that directly or indirectly affect growth hormone (Fr N 2266516, 1975; Fr N 2532308, 1983; EP N 0143850, 1984; PCT 89/0711, 1989; PCT N 90 / 13570, 1990; PCN / 16923, 1991; US N 5008244, 1991).

 It is known, however, that growth hormone does not exhibit strict specificity, simultaneously affects many metabolic pathways and, in effective doses, has a broad therapeutic effect (US N 4816439, 1989; PCT 90/12589, 1990; PCT 90/14839, 1990 US N 5006510, 1991; PCT 91/15227, 1991).

 To date, two peptide preparations are known. One of them has a specific focus on stimulating the development of the epidermal layer of the skin in humans (PCT N 90/13570, 1990), the other has a metal peptide, pronounced specificity for hair growth in warm-blooded animals (PCT N 91/07431, 1991).

 However, the known methods for producing biologically active peptides are complex, time-consuming, the product yield is low, and the use of known agents is mostly accompanied by undesirable side effects.

The present invention posed and solved the problem of creating an effective way to obtain a new, previously unknown biologically active peptide of the formula:
H-Arg-Tyr-D-Ala-Phe-Gly-A,
where A is OH or a substituted amide (C ₁ - C ₃ ),
having a specific, stimulating effect on the activity of the growth zone of the epithelium, the development of the epidermal layer, hair growth, and also affecting the increase in live weight.

 The essence of the new method consists in the synthesis of a peptide in a solution by sequentially chain extension from the C-terminus of the molecule, using the strategy of maximum blocking of functional groups, starting from glycine tert-butyl ether using the activated esters method and the mixed anhydrides method using carbobenzoxy and tert-butyloxycarbonyl amino acids.

The method is illustrated by the following example:
Example 1. H-Arg-Tyr-D-Ala-Phe-Gly-OH
Obtaining BOC-Arg (BOC) -Tyr (OBut) -D-Ala-Phe-Gly-oBut.

1. Z-Phe-Gly-oBut
46.5 g (0.1 mol) of Z-Phe-OPfP was dissolved in 150 ml of dimethylformamide and a solution of 21.3 g of glycine phosphate tert-butyl ether and 11.5 ml of N-methylmorpholine in aqueous dimethylformamide was added with stirring. The reaction mixture was stirred for 24 hours at room temperature. Dimethylformamide was distilled off in vacuo, the residue was poured with 200 ml of ethyl acetate and washed with 2% sulfuric acid 2 times 150 ml, then washed with saturated sodium bicarbonate 2 times 150 ml, washed with water to a neutral medium, dried over anhydrous sodium sulfate. The ethyl acetate layer was evaporated in vacuo. The residue was crystallized from ether with hexane. Yield: 37.3 g (90.5%). R _f = 0.68 (system: chloroform: ethyl acetate: methanol = 3: 6: 1), R _f = 0.59 (system: ethyl acetate: hexane = 1: 1).

2. ZD-Ala-Phe-Gly-oBut
37.3 g (0.09 mol) of Z-Phe-Gly-oBut was dissolved in 200 ml of ethyl alcohol, 4.0 g of palladium catalyst was added and a stream of hydrogen was passed for 4 hours. The catalyst was filtered off, the solvents were evaporated in vacuo to a thick oil. up to constant weight.

34.4 g (0.1 mol) of ZD-Ala-ONP was dissolved in 100 ml of dimethylformamide and a solution of 13.5 g (0.1 mol) of hydroxybenzotriazole and 25.2 g (0.09 mol) of H-Phe were added with stirring -Gly-oBut in 70 ml of dimethylformamide. The reaction mixture was stirred at 20 ^o C. for a day. Dimethylformamide was distilled off in vacuo, the residue was poured with 200 ml of ethyl acetate and washed with 2% sulfuric acid 2 times 100 ml, then washed with 2% ammonia 4 times 80 ml, washed with water until neutral, dried over anhydrous sodium sulfate. The ethyl acetate layer was evaporated in vacuo. The residue was crystallized from ether. Yield: 39.5 g (90.8%). R _f - 0.76 (system: chloroform: ethyl acetate: methanol = 6: 3: 1), R _f = 0.52 (system: chloroform: methanol = 9: 1).

3. Z-Tyr (oBut) -D-Ala-Phe-Gly-OBut
39.5 g (0.08 mol) of ZD-Ala-Phe-Gly-oBut was dissolved in 200 ml of ethyl alcohol, 4.0 g of palladium catalyst was added and a stream of hydrogen was passed for 4 hours. The catalyst was filtered off, the solvents were evaporated in vacuo to thick oil, to constant weight.

34.9 g (0.09 mol) of Z-Tyr (oBut) -Oh and 10.3 ml (0.09 mmol) of N-methylmorpholine in 150 ml of dimethylformamide were cooled to -15 ^° C and 12.6 g was added with stirring (0.09 mol) of isobutyl chloroformate. After 2 minutes, a cooled solution of 28.6 g (0.08 mol) of the tripeptide tert-butyl ether of the tripeptide: HD-Ala-Phe-Gly-oBut was added. The reaction mixture was stirred at -15 ^° C. for 30 minutes and 2 hours at room temperature. Dimethylformamide was distilled off in vacuo, the residue was poured with 200 ml of ethyl acetate and extracted with 2% sulfuric acid 2 times 80 ml, then washed with saturated sodium bicarbonate 2 times 80 ml, washed with water to a neutral medium, dried over anhydrous sodium sulfate. The ethyl acetate layer was evaporated in vacuo. The residue was crystallized from ethyl acetate. Yield: 51.3 g (95.4%), R _f = 0.61 (system: chloroform: ethyl acetate: methanol = 6: 3: 1), R _f = 0.55 (system: chloroform: methanol = 9: one).

4. BOC-Arg (BOC) -Tyr (oBut) -D-Ala-Phe-Gly-OBut
51.3 g (0.07 mol) of Z-Tyr- (BOC) -D-Ala-Phe-Gly-OBut solvents in 200 ml of ethyl alcohol, 4.0 g of palladium catalyst was added and a stream of hydrogen was passed for 4 hours. The catalyst was filtered off, dissolved, evaporated in vacuo to a thick oil, to constant weight.

42.0 g (0.07 mol) of H-Tyr (OBut) -D-Ala-Phe-Gly-OBut was dissolved in 150 ml of dimethylformamide and 9.2 ml (0.08 mol) of N-methylmorpholine, 30.0 g (0.08 mol) of BOC-Arg (BOC) -OH and 10.8 g of oxybenzotriazole. The reaction mixture was cooled with stirring to -10 ^° C and 16.5 g (0.08 mol) of dicyclohexylcarbodiimide were added. The reaction mixture was stirred for 3 days at room temperature. Dicyclohexylurea was filtered off, the solvent was distilled off in vacuo, the residue was poured with 300 ml of ethyl acetate and washed with 2% sulfuric acid 2 times 150 ml, then washed with saturated sodium bicarbonate 2 times 100 ml, washed with water to a neutral medium, dried over anhydrous sulfate sodium. The ethyl acetate layer was evaporated in vacuo. The residue was crystallized from ethyl acetate. Yield: 62.6 g (93.5%), R _f = 0.71 (system: chloroform: ethyl acetate: methanol: acetic acid = 6: 3: 1: 0.5), R _f = 0.22 (system: chloroform : methanol = 9: 1).

5. Obtaining H-Arg-Tyr-D-Ala-Phe-Gly-OH
62.6 g (0.065 mol) of BOC-Arg (BOC) -Tyr (OBut) -D-Ala-Phe-Gly-OBut was dissolved in 80 ml of 50% trifluoroacetic acid in chloroform. After 1 h, the solvents were evaporated in vacuo to a thick oil. The residue was poured with ether and rubbed. The precipitate was filtered off and washed with ether, dried in air. Yield: 39.8 g (100%), R _f = 0.44 (system: butanol: acetic acid: water = 3: 1: 1).

 The peptide was purified by ion exchange chromatography on a Sephadex column in a gradient of 0.1-1.0 M pyridine acetate buffer. Yield: 35.8 g (90.0%).

As a result of studying the physicochemical properties of the new peptide, the following characteristics were obtained:
Primary structure - H-Arg-Tyr-D-Ala-Phe-Gly-OH
Gross formula - C ₂₉ H ₄₀ N ₈ O ₇
Molecular Weight - 612.6 Da
Appearance - white with yellowish or gray powder
Solvent - soluble in water, 0.1% aqueous solution is transparent, sparingly soluble in alcohol, practically insoluble in chloroform
UV spectrum - a characteristic absorption maximum of a 0.02% aqueous solution of 275 ± 2 nm, a shoulder of 282 ± 2 nm.

 As a result of studying the biological activity of the new peptide, data were obtained that testify to its effect on physiological processes such as: an increase in body weight; activation of the growth zone of the epithelium, stimulation of the development of the epidermis and hairline in various animal species.

 The effect of peptide preparation on weight gain.

 A. The experiment was carried out on 4 groups of outbred white rat pups at the age of one month. Control group 1 received injections of saline, 2,3 and 4 experimental groups were administered the drug in doses of 0.1; 0.5; 1.0 μg / kg of weight intraperitoneally. Rats were weighed in all groups every 5 days and the amount of food consumed was recorded in parallel. The experiment lasted for 35 days. The results are shown in FIG. 1. The highest weight gain was noted in the group receiving 1.0 μg / kg of peptide. Data showing the amount of food consumed by rats is shown in FIG. 2. In all experimental groups, there was no effect of the peptide on the amount of food consumed, which indicates its anabolic activity.

 B. The experiment was conducted on 7 groups of NMRI mice in a free diet with unlimited access to water and food. At the same time, group 1 received injections of saline, groups 2, 3, and 4 of mice received a peptide along with drink at the rate of 1.0, 10.0, and 100 μg / kg, respectively, groups 5, 6, and 7 peptide were administered daily intraperitoneally at doses of 0, 1, 0.5 and 1.0 μg / kg, respectively. The weight of the mice was determined twice a week for 30 days and the amount of feed consumed was recorded daily. The total results of the change in the weight of mice with the described methods for administering the peptide are presented in FIG. 3. The most effective is taking the peptide with a drink at a dose of 1 μg / kg (p / o).

 In FIG. 4 presents the results of the number of average daily food intake in different groups. Statically significantly (p <0.05) more food was consumed by mice that received the drug with a drink at a dose of 1.0 μg / kg orally, i.e. animals that gave maximum weight gain.

 Given that under the influence of the peptide, two parameters change: weight gain and food consumption, weight gain is calculated as a percentage when 1 g of food is consumed, which is an indicator of the commercial benefit of using the peptide. In FIG. 5 presents the results. It can be seen that the largest weight gain with the consumption of 1 g of food was noted when taking the peptide with a drink at a dose of 10.0 μg / kg, which is 36.5% higher than the gain in the control group.

 The effect of the peptide on the morphological changes in the skin and hairline growth.

 The experiment was performed on 4 groups of NMRI mice: group 1 - passive control, groups 2, 3, and 4 received the peptide preparation orally at doses of 1.0, 10.0, and 100 μg / kg, respectively, when drinking. After a biopsy, skin samples taken at the withers area of 5x5 mm were fixed by the Lilly method for 48 hours, washed with a descending battery in alcohols and immersed in Hemolown Meyer dye for 24 hours. The preparation of histological preparations was carried out using a Reichard microtome, orienting pieces of skin in the plane of the axial hair, the sections were stained using the classical eosin technique.

 In the study of histological preparations with a thickness of 10 mm, the qualitative state of the epidermis and dermis was evaluated, and the number of hair follicles of 1 mm was determined by the Aftandilov method in experimental and control groups of mice. The results are shown in FIG. 6. From the diagrams in the figure it is seen that in mice receiving 100, 10.0, 1.0 μg / kg of peptide, an increase in the number of hair follicles by 14, 19 and 22%, respectively, was observed compared with the control group.

 In these groups of mice, the length of the hair at the withers was evaluated. Up to a micron, each mouse measured the length of 10 hairs and calculated the average values for each group. The results are shown in FIG. 6, 7.

 The study of the toxicity of the drug.

 Studies of the possible toxicity of the new peptide for the determination of LD-50 were carried out on 24 healthy mongrel white mice weighing 18 g, which were in the same conditions of nutrition and maintenance. In all groups, half of the animals were males and the other females. 24 hours before the tests and during their conduct, the animals were in a vivarium with constant temperature and ventilation. 2 hours before the start of the test, the animals were cut off water and food.

 Four groups of mice, 8 animals in each group, were isolated. In this case, 1, 2 and 3 groups of mice were injected with an aqueous solution of the drug at a dose of 800, 1100 and 1400 mg / kg, respectively. Mice of the 4th group, control, were injected with physiological saline in the same volume. Observations of animals were carried out for 7 days after administration. It was found that immediately after administration and during the entire observation period, no changes in the general condition and behavior of the animals were observed. No cases of animal mortality were also noted during this period.

 The results of the studies showed that when administered intraperitoneally, the peptide at a dose of 800, 1100 and 1400 mg / kg does not have a toxic effect; at these doses, it was not possible to achieve a dose of LD-50.

 Thus, the new peptide of the formula: H-Arg-Tyr-D-Ala-Phe-Gly-OH is not toxic and has a pronounced stimulating effect on the activity of the growth zone of the epithelium, on the development of the epidermal layer of the skin, and contributes to an increase in the density of hair follicles and hair growth It also affects the increase in body weight.

Claims (1)

A method of producing a peptide having anabolic activity, stimulating weight gain, the development of the epidermal layer and hairline growth, which consists in introducing glycine tert-butyl ether with benzyloxycarbonylphenylalanine in a solution, and then the peptide chain is gradually increased by the method of activated esters and the mixed anhydride method, carbobenzoxy amino acids are sequentially attached with preliminary cleavage at each stage of the carbobenzoxy group by processing the reaction ion mixture by a stream of hydrogen in the presence of a palladium catalyst and subsequent purification of the intermediate products by crystallization, finally release in an acidic medium and the target product of the formula
H - Arg - Tyr - D - Ala - Phe - Gly - A,
where A is OH or substituted C ₁ - C ₃ amide,
purified by ion exchange chromatography.

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