JP6552960B2 - Methods for producing recombinant peptides and resulting peptides - Google Patents

Description

Cross-reference to related applications
This application claims priority to Russian Federation Patent Application No. 2012111965, filed March 28, 2012, the United States of International Patent Application No. PCT / RU2013 / 000433 filed May 28, 2013 It is a US domestic phased application under 351 Patent Act. The international application was published on October 10, 2013 as International Publication No. WO2013 / 151467 based on Article 21 (2) of the Patent Cooperation Treaty. The entire contents of these applications are incorporated herein by reference.

Sequence listing
This application contains a Sequence Listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The above ASCII copy created on October 31, 2014 is called 246273.000002_SL.txt and has a size of 503,566 bytes.

FIELD OF THE INVENTION The present invention relates to the field of biochemistry, to recombinant methods for the production of peptides and to the resulting peptides. In particular, the present invention provides the following general formula:
A-Thr-Lys-Pro-BCDX
(In the formula,
A-0, Met, Met (O), Thr, Ala, His, Phe, Lys, Gly
B-0, Gly, Asp, Trp, Gln, Asn, Tyr, Pro, Arg
C-0, Arg, Phe, Tyr, Gly, His, Pro, Lys
D-0, Val, Gly, Tyr, Trp, Phe, His
X-OH, OCH ₃ , NH ₂
Where 0 is no amino acid residue. However, when A ≠ 0, B and / or C and / or D ≠ 0, and when B ≠ 0, C and / or D ≠ 0, and Phe-Thr-Lys-Pro-Gly, Thr- Excludes Lys-Pro-Pro-Arg and Thr-Lys-Pro-Arg-Gly peptides. )
It relates to a peptide having

Background of the Invention Small peptides are known to exhibit very high activity and promote spontaneous healing in damaged organs. As is generally known, smaller peptides are initially derived from animal tissue and later produced under laboratory conditions. Drugs containing peptides are capable of repairing damaged somatic cells and restoring and restoring lost function to affected organs. Drugs containing peptides have been developed over 30 years ago, and since then hundreds of experiments have been conducted to treat and prevent various systemic diseases, and the effectiveness of peptides in rejuvenation of individual organs and whole bodies Sex has been proven.

In the body, peptides serve as information messengers. The peptide transmits information from one cell to another so that everything can be done on time and on time. If one cell functions properly, the corresponding organ works well. When dysfunction occurs, it affects the entire organ and causes disease. Obviously, it is possible to treat the disease by introducing a deficient substance into the body, but this approach completely “damages” the cells and stops the proper functioning. There is a risk that. Thus, there is a need to deliver to the cells peptide messengers that cause the cells to function in a way that the body heals spontaneously. Each organ has a supply of storage stem cells. If this supply is consumed evenly, one can live to 100-110 years old. The peptide is the same for all mammals. Thus, when a calf peptide is introduced into humans, the human body incorporates it as a native molecule. The main task was to find a way to isolate the peptide from animal organs. This technology was invented as early as 1971 by professors Vladimir Morozov and Vyacheslav Havinson of the military hospital. Medicaments were developed and based on them, nutraceuticals were made for ease of use. In a study of the aging process and how it affects aging, researchers at the St. Petersburg Geriatric Institute concluded that the average life span was increased by 30-38% when peptides were introduced into the experimental group of mice. Later, peptides were studied for the elderly in the Kiev Geriatric Institute and St. Petersburg. This showed almost a two-fold reduction in mortality, suggesting a high senile protective activity of the peptide. Although long-term studies and use of peptide drugs have shown these high efficacy in patients of different age groups, special efficacy has been observed in the elderly (over 50 years). The absolute advantage of peptide biomodulators is that there are no side effects. Have various medical conditions within 26 years 1
More than 5 million people have accepted such products. Their effectiveness averaged up to 75-95%. The peptide deficiency that occurs with age and disease changes significantly accelerates tissue wasting and body aging. In fact, the proper amount of peptide is required for proper functioning of cells and tissues, and the proper amount of peptide also supports optimal gene function. Peptides that are functional in cells specific for the peptide are synthesized in the cells. Thus, in disease state changes, and with age, cellular function is destroyed, so peptide replication is also affected. As a result, cell function is affected secondarily. Thus, tissue degeneration progresses and eventually appears clinically. Therefore, the application of low molecular weight peptides is one of the major innovations in medicine, which can significantly slow the rate of aging by stimulating cell proliferation and tissue regeneration and improving cell life. Another important advantage of peptides is their anti-tumor activity. Currently, the use of peptides is the best uncompromising solution in activation (rejuvenation of the body) and cancer prevention. Because it is not only by the regulation and synchronization of all circulation processes, but also by increasing cell division capacity without atypical (atypical is the wrong cell structure, or abnormal), cells and tissues To rejuvenate the

  Of course, at the current stage of the technology, production of low molecular weight peptides from animal tissue is impractical. The reason is that this method is very expensive although it is needless to say that the humanity of the production process is not.

  Modern and advanced methods of production involve the use of recombinant microorganisms. The most convenient microorganism is E. coli. Commercial strains of E. coli suitable for use under laboratory conditions, such as E. coli K12, E. coli O104, are known in the prior art. These and similar known E. coli strains can be used to obtain strains that produce the claimed low molecular weight peptides. The nucleic acid encoding the corresponding target small molecule peptide can be incorporated into any known strain suitable for use under laboratory conditions. Vectors such as bacterial plasmids, viruses, virions, hybrid vectors containing phage DNA, and plasmids are used to insert DNA into host cells. These vectors include, for example, cosmid and phasmid.

Furthermore, it is possible to produce peptides by conventional chemical synthesis.
The proposed peptide solves the still important issue of expanding the range of approaches for stimulating reproduction and sexual function and treating reproductive and sexual dysfunction. Currently, pathogenic treatments for such conditions are performed using psychotherapy, antidepressants, anti-anxiety agents, adaptogens, and vitamins, general factory stimulants, and diet supplements. Such treatment is long, inefficient and has many side effects.

  One of the most effective class of physiologically active substances suitable for the production of stimulants of reproductive and sexual function is peptides, which as endogenous substances have virtually no negative side effects.

Our own research has shown that Selank, a heptapeptide of the general formula Thr-Lys-Pro-Arg-Pro-Gly-Pro, which is a synthetic analogue of taftsin which is an endogenously produced peptide, is reproductive and sexual. It has been shown that it can be used as an approach for the treatment and prevention of dysfunction (Russian Federal Patent No. 2404793). However, the synthesis of Selank, which is a heptapeptide, is multi-step, and the cost of pharmaceuticals based on this is greatly increased. Furthermore, exposure to strong proteolysis in the body reduces its stimulatory effect on the prevention and treatment of reproductive and sexual dysfunction.

DISCLOSURE OF THE INVENTION The object of the invention is to expand the scope of approaches having reproductive and sexual function stimulating activity.

  The technical outcome achieved when practicing the present invention is increased effectiveness of prevention and treatment of reproductive and sexual dysfunction, shortening the duration of the course of therapy, and reducing the cost of the drug. Here, a peptide having the following general formula is proposed as a pharmaceutical.

A-Thr-Lys-Pro-BCDX
(In the formula,
A-0, Met, Met (O), Thr, Ala, His, Phe, Lys, Gly
B-0, Gly, Asp, Trp, Gln, Asn, Tyr, Pro, Arg
C-0, Arg, Phe, Tyr, Gly, His, Pro, Lys
D-0, Val, Gly, Tyr, Trp, Phe, His
X-OH, OCH ₃ , NH ₂
Where 0 is no amino acid residue.
However, when A ≠ 0, B and / or C and / or D ≠ 0, and when B ≠ 0, C and / or D ≠ 0, and Phe-Thr-Lys-Pro-Gly, Thr- Excludes Lys-Pro-Pro-Arg and Thr-Lys-Pro-Arg-Gly peptides. )

The selection of amino acid residues at positions A, B, C, D, and X is based on the N-terminal and C-terminal amino acid residues in the database [EROP-Moscow (http://erop.inbi.ras.ru/) Zamyatin AA]. Based on bioinformatic analysis of amino acid residue frequency at corresponding positions of groups. The selection of these amino acid residues was based on a criterion for the appearance rate exceeding 50% of the amino acid residues at that position. Samples of this amino acid residue were confirmed experimentally by synthesizing individual peptides and testing them for stimulation of reproductive and sexual function activity in in vivo models. The pharmacophore position in the peptide is determined by experiment. All peptides are known to be exposed to peptidases and break down into specific fragments. For this purpose, Selank of Thr-Lys, Thr-Lys-Pro, Pro-Gly-Pro, Arg-Pro-Gly-Pro, Pro-Arg-Pro-Gly-Pro
Heptapeptide fragments were synthesized and their activities were studied. The results are shown in Table 4. In this analysis, the pharmacophore was determined to be Thr-Lys-Pro. The binding of the experimentally identified individual amino acid residues to the C-terminus has been shown to maintain the activity of the peptide (stimulating reproductive and sexual function). However, as shown by studies (Table 5, Example 10), the number of amino acid residues in the peptide is 3 or greater than 4. Tetrapeptides have no reproductive and sexual function stimulating activity.

The above technical result directly synthesizes various peptides having the general formula A-Thr-Lys-Pro-BCDX (except tetrapeptides), and prevents and treats these peptides for reproductive and sexual dysfunction. Achieved by using it as a reproductive and sexual function stimulant. Our own research shows that as a stimulator of reproductive and sexual function, a synthesized peptide corresponding to the general formula A-Thr-Lys-Pro-BCDX, namely Thr-Lys-Pro tripeptide, Thr-Lys- It was shown that Pro-Arg-Pro pentapeptide and Thr-Lys-Pro-Arg-Pro-Phe hexapeptide can be recommended.

All peptides with A-Thr-Lys-Pro-BCDX (except tetrapeptides) recommended as stimulators of reproduction and sexual function have a common pattern. That is, there are Thr-Lys-Pro tripeptide molecules in their molecular structure.

Thr-Lys-Pro-Arg-Pro-Gly-Pro heptapeptide (Selank) was used as a control.

The outcome of the studies carried out is that peptides having the general formula A-Thr-Lys-Pro-BCDX (except for tetrapeptides) have reproductive and sexual function stimulating activity and prevent and treat reproductive and sexual dysfunction It can be used as a medicine for General Formula A-Thr-Lys-Pro-BCDX
Table 1 shows some of the peptides.

FIG. 5 is a diagram of Arg-Pro-Gly-Pro tetrapeptide synthesis. FIG. 1 is a diagram of Pro-Arg-Pro-Gly-Pro pentapeptide synthesis. FIG. 1 is a diagram of Pro-Gly-Pro tripeptide synthesis. FIG. 5 is a diagram of Thr-Lys-Pro-Arg-Pro-Phe hexapeptide synthesis. FIG. 5 is a diagram of Thr-Lys-Pro-Arg-Pro pentapeptide synthesis. FIG. 5 is a diagram of Thr-Lys dipeptide synthesis. FIG. 4 is a diagram of Thr-Lys-Pro-Phe tetrapeptide synthesis.

BEST MODE FOR CARRYING OUT THE INVENTION The following are examples illustrating the invention.

A peptide having the general formula A-Thr-Lys-Pro-BCDX was synthesized by peptide chemistry in solution using L amino acids. Perform peptide synthesis by sequential extension of the peptide chain and fragment condensation using mixed anhydride method, carbodiimide method with 1-hydroxybenzotriazole added as conucleophile, activated ester method, and mixed anhydride method did. All intermediate and final products were isolated and characterized. Evaporation of the solution was carried out at 40 ° C. using a vacuum evaporator. The melting point determined with the Boethius apparatus is shown without calibration. Silufol silica gel coated plate (Czech Republic)
Tested by TLC above. Under a chlorine environment, substances were detected by UV light using ninhydrin, Burton reagent, Pauly reagent, Reindel-Hoppe reagent, and o-tolidine. The specific rotation was obtained by an AI-EPO polarimeter. Peptide homogeneity was examined by high performance liquid chromatography (HPLC) and peptide structure was confirmed by mass spectrometry. All solvents were correspondingly dehydrated. Melting points are not calibrated.

Furthermore, peptides were obtained by genetic engineering techniques using host cells designed with known laboratory strains of E. coli. These E. coli strains were transformed with a known commercial plasmid containing a nucleic acid encoding the target peptide.

  In the examples, peptide synthesis is described.

Synthesis of Arg-Pro-Gly-Pro tetrapeptide The tetrapeptide was synthesized according to the diagram shown in FIG.

The mixed anhydride method, azide method, and carbodiimide method were used for the synthesis. Derivatives of L amino acids were used for the synthesis. Evaporation of the solution was carried out at 40 ° C. using a rotary evaporator. The melting point determined on the Boethius device is shown without calibration. The identity of the resulting compounds was tested by TLC on Silufol silica gel coated plates (Czech Republic). Substances were detected by spraying a solution of ninhydrin and / or o-tolidine onto the plate. Chromatographic transfer rate (Rf) values in the following solvent systems are shown. Acetone: benzene: acetic acid (50: 100: 1)-(1); chloroform: methanol (9: 1)-(2); hexane: acetone (3: 2)-(3); butanol: acetic acid: water (4 Butanol: acetic acid: pyridine: water (30: 6: 20: 24)-(5); chloroform: methanol: ammonia (6: 4: 1)-(6); benzene: Ethyl acetate: acetone: 50% acetic acid: water (2: 1: 1)-(8); chloroform: methanol (14: 1)-(9); chloroform: methanol: Ammonia (8: 1.75: 0.25)-(10); chloroform: methanol: ammonia (6.5: 3.0: 0.5)-(11).

The specific rotation was obtained by an AI-EPO polarimeter.
Elemental analysis was performed using a Carlo-Erba model 1106 analyzer.

I. Boc-Pro-Gly-OEt. 8.3 g (3.45 mmol) of Boc-Pro-OH was dissolved in 50 ml of CH ₂ Cl ₂ and cooled to 5 ° C. After that, 38.45 mmol (5.38 ml) of TEA was added. The reaction mixture was cooled to -25 to -30.degree. At this temperature, 38.45 mmol (4.84 ml) isobutyl chloroformate was added using a pipette. The reaction mixture temperature was maintained in the range of -18 to -20.degree. C. for 20 minutes. At the same time, a solution of 5.9 g (42.3 mmol) 1.1-fold excess of HCl.H-Gly-OEt in 75 ml chloroform containing 5.92 ml TEA. The solution was cooled to -25.degree. C. and after formation of the mixed anhydride in the first flask, the contents were poured immediately into ether solution. The reaction mixture was incubated at -10.degree. C. for 1 hour and then stirred with a magnetic stirrer at 4.degree. C. for 12 hours. The reaction mixture is evaporated and then 250 ml of ethyl acetate are added. The ethyl acetate solution was then washed three times with 25 ml of 0.1 N HCl, three times with 25 ml of H ₂ O and once with a saturated solution of NaCl. The organic layer was dried over MgSO ₄ , filtered and evaporated. The residue was dried under vacuum with P ₂ O ₅ / KOH and paraffin.
Yield: 10.2 g (30.3 mmol) 78.83%
Rf 0.5 (7); 0.862 (8)
Melting point 68-70 ° C

II. Boc-Pro-Gly- N 2 H 3. 10.2g of Boc-Pro-Gly-OEt the (30.3 mmol) was dissolved in anhydrous methanol 80 ml, 4-fold excess, i.e., 5.88ml (121. 2 mmol) of hydrazine hydrate were added. The solution was stirred at room temperature with a magnetic stirrer for 12 hours. The reaction mixture was evaporated and then twice with ether. The residue was then poured into ether (̃5 ml) and left in the refrigerator overnight (seeding agent added for better crystallization). The precipitated crystals were filtered, washed with ether using a filter and dried in a desiccator.
Yield: 6.9 g (20.79 mmol) 68.62%
Rf 0.284 (7); 0.474 (8); 0.189 (9)
Melting point 98-100 ° C

III. Boc-Pro-Gly-Pro-OBzl. A solution of Boc-Pro-Gly-N ₂ H ₃ containing 4.7 g (14.6 mmol) in 40 ml of DMF was added to 58.4 mmol of ethyl acetate ( (4-fold excess) of hydrogen chloride was added, cooled to -20 ° C, and 1.73 mL (14.6 mmol) of freshly distilled tert-butyl nitrite was added immediately. The reaction mixture was then stirred at -5 ° C for 30 minutes. The reaction mixture was cooled to −40 ° C. and a solution of 8.2 mL (58.4 mmol) of TEA in 4 mL of DMF cooled to −10 ° C. was added. When the temperature of the reaction mixture rose to −20 ° C., 3.7 g (15.3 mmol) of 1.05 fold excess of HCl.H-Pro-OBzl was added to 20 ml of DMF and 2.14 ml of TEA. Thereafter, it was stirred at 4 ° C. for 24 hours with a magnetic stirrer. The reaction mixture was evaporated and the residue dissolved in ethyl acetate 200 mL, 2 times with _{H 2} O in 20ml, 3 times with 10% sodium KHSO ₄ of 20ml, 3 times with _{H 2} O in 20ml, 5% NaHCO of 20ml Washed 3 times with ₃ and 3 times with 20 ml of H ₂ O. The ethyl acetate solution was dried by MgSO ₄ and. Then it was evaporated and a small amount of ether (-10 mL) was added to the residue. Then, it was left to stand in a refrigerator for crystallization. A seeding agent was added for better product crystallization. The precipitated crystals were filtered and washed with a small amount of ether using a filter. It was then dried in a desiccator.
Yield: 5.358 g (11.65 mmol) 79.92%
Rf 0.326 (7); 0.947 (8); 0.390 (9)
Melting point 125-126 ° C
[Α] _D ²² = −101.18 ° (c = 0.85; CH ₃ OH)
Elemental analysis: С62.89 (62.73); N9.21 (9.14); H7.52 (7.24)

IV. TFA.H -Pro-Gly-Pro-OBzl. 5.358 g (11.65 mmol) of Boc-Pro-Gly-Pro-OBzl was dissolved in 29.13 ml of methylene chloride. Then 29.13 mL of TFA was added, incubated at room temperature for 45 minutes, evaporated twice with absolute ethanol, twice with benzene and twice with ether and dissolved in benzene. Then hexane was poured on. Hexane was decanted and the resulting material was dried under vacuum in a desiccator with KOH, P ₂ O ₅ and paraffin while the desiccant was changed several times.
Yield: 4.63 g (9.7 mmol) 98%
Rf 0.043 (7); 0.247 (8); 0.018 (9)

V. Boc-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl.
3.09 g of Boc-Arg (NO ₂ ) —OH (9.7 mmol) in 50 mL THF and 10 ml
Dissolved in DMF, added 2.07 g (10.67 mmol) of DCC, cooled to 0 ° C. and stirred for 40 minutes. Then, a solution of TFA · H-Pro-Gly-Pro-OBzl was added to 50 ml of THF and 4.46 ml (9.7 mmol) of TEA. The reaction mixture was stirred for 3 days. The DCM precipitate was filtered off and the solution was evaporated under vacuum, after which 200 ml of hexane was added to the residue. This is used to separate the desired product as an oil, which is dissolved in 500 mL of ethyl acetate, 3 times with 25 ml 0.1 N HCl, 3 times with 25 ml H ₂ O, 1 with a saturated solution of NaCl. Washed twice. The organic layer was dried over MgSO ₄ , filtered and evaporated. The residue was dissolved in ethyl acetate and precipitated with dry ether. The precipitate was filtered off and dried under vacuum with P ₂ O ₅ / KOH and paraffin, while changing the desiccant several times.
Yield: 4.76 g (7.9 mmol) 85%
Rf 0.44 (1); 0.8 (11)
Melting point 108 to 110 ° C
[Α] _D ²² = -77.8 ° (c = 0.5; CH ₃ COOH)

VI. Was dissolved Boc-Arg-Pro-Gly- Pro. Boc-Arg (NO 2) of 4.76g (6.5mmol) -Pro-Gly- Pro-OBzl in methanol 100 ml, then, 1 ml 1N hydrochloric acid and 4.67
g catalyst, ie 10% palladium oxide on neutral aluminum oxide,
Hydrogenation was carried out for 6 hours while flowing dry hydrogen at room temperature under m. The catalyst was then filtered off and washed on the filter with methanol. The pooled filtrate was evaporated to dryness. The residue was precipitated from anhydrous methanol with ether. Then, it was made to dry under vacuum and during that time, the desiccant was changed several times.
Yield: 4.23 g (5.8 mmol) 89%
Rf 0.125 (4); 0.57 (6); 0.37 (5)
Melting point 123-125 ° C

VII. Arg-Pro-Gly-Pro. 4.23 g (5.8 mmol) of Boc-Arg-Pro-Gly-Pro-OH were suspended in 10 ml of 2N hydrochloric acid in dioxane and incubated for 45 minutes at room temperature. Thereafter, dry ether was added and the precipitate was washed by dry ether. It was reprecipitated from anhydrous methanol with ether. The resulting precipitate was dissolved in 30% ethanol 7.5ml, Amberlyst A-21 for the acetic acid / hydrochloride salt exchange (AcO ^- form) was applied to the column.
The peptide was eluted with 200 ml of 30% ethanol, evaporated to dryness under vacuum and precipitated from methanol with anhydrous ether.
Yield: 3.69 g (4.93 mmol) 85%
Rf 0.287 (4); 0.145 (5); 0.338 (14)
Melting point 120-122 ° C
HPLC result: Column: Supercosil ABZ Plus, size 4.6 × 250 mm, flow rate
1 mL / min, solvent A: NH ₄ H ₂ PO ₄ + H ₃ PO ₄ (50 mM, pH 2.8), solvent B: MeOH
Gradient: 0 to 20 minutes (0 to 40% B), retention time 21.21 minutes Rf 0.24 (6)
Melting point 151 ° C
[Α] _D ²² = -65.0 ° (c = 0.5 CH ₃ COOH)
HPLC result: Column: Zorbax ODS d = 4.6 mm; t = 35 ° C.
Flow rate 1 mL / min; A = 50 mM NH ₄ H ₂ PO ₄ (pH 2.5); B = A + MeOH (1: 1); 10-60% B (within 25 minutes)
Retention time 16.5 minutes

Synthesis of Pro-Arg-Pro-Gly-Pro Pentapeptide Peptide synthesis was carried out by the classical method of peptide chemistry using native L amino acids according to the diagram shown in FIG.

Initially, Pro-Gly-Pro tripeptide was produced, and then pentapeptide was obtained by stepwise deposition of peptide chains from the N-terminus. In the synthesis, a mixed anhydride method, an azide method and a carbodiimide method were used.

  The chromatographic transfer rate (Rl) values in the following solvent systems are indicated. Butanol: acetic acid: water (4: 1: 1)-(1); chloroform: methanol: ammonia (6: 4: 1)-(2); acetone: benzene: acetic acid (50: 100: 1)-(3) Chloroform: methanol (9: 1)-(4); hexane: acetone (3: 2)-(5); butanol: acetic acid: pyridine: water (30: 6: 20: 24)-(6); chloroform: Methanol (14: 1)-(7).

I. Boc-Pro-Gly-OEt. 8.3 g Boc-Pro (38.45 mmol) was dissolved in 50 ml CH ₂ Cl ₂ , cooled to + 5 ° C. and 38.45 mmol (5.38 ml) TEA was added. Added. The reaction mixture was cooled to -25 to -30.degree. At this temperature, 38.45 mmol (4.84 ml) isobutyl chloroformate was added using a pipette. The reaction mixture temperature was maintained in the range of −18 to −20 ° C. for 20 minutes. At the same time, a solution of 5.9 g (42.3 mmol) 1.1-fold excess of HCl.H-Gly-OEt in 75 ml chloroform containing 5.92 ml TEA. The solution was cooled to -25.degree. C. and after formation of the mixed anhydride in the first flask, the contents were immediately poured into ether solution. The reaction mixture was incubated at −10 ° C. for 1 hour and then stirred with a magnetic stirrer at 4 ° C. for 12 hours. The reaction mixture was evaporated and 250 ml of ethyl acetate was added. The ethyl acetate solution was washed 3 times with 25 ml 0.1 N HCl, 3 times with 25 ml H ₂ O and once with a saturated solution of NaCl. The organic layer was dried over MgSO ₄ , filtered and evaporated. The residue was dried under vacuum with P ₂ O ₅ / KOH and paraffin.
Yield: 10.2 g (30.3 mmol) 78.83%
Rf 0.5 (3); 0.862 (4)

II. Boc-Pro-Gly- N 2 H 3. 10.2g of Boc-Pro-Gly-OEt the (30.3 mmol) was dissolved in anhydrous methanol 80 ml, 4-fold excess of hydrazine hydrate, i.e., 5. 88 ml (121.2 mmol) were added. The solution was stirred with a magnetic stirrer for 12 hours at room temperature. The reaction mixture was evaporated and then twice with ether, then ether (-5 ml) was poured on the residue and left in the refrigerator overnight (seeding agent was added for better crystallization). The precipitated crystals were filtered, washed with ether using a filter and dried in a desiccator.
Yield: 6.9 g (20.79 mmol) 68.62%
Melting point 98-100 ° C
Rf 0.284 (3); 0.474 (4); 0.189 (5)

III. Boc-Pro-Gly-Pro-OBzl.
Boc-Pro-Gly-N ₂ H containing 4.7 g (14.6 mmol) in 40 ml DMF, cooled to −20 ° C., 58.4 mmol (4-fold excess) hydrogen chloride in ethyl acetate. _To the solution of ₃ , 1.73 mL (14.6 mmol) freshly distilled tert-butyl nitrite was added immediately. The reaction mixture was then stirred at -5 ° C for 30 minutes. The reaction mixture was cooled to −40 ° C. and a solution of 8.2 mL (58.4 mmol) of TEA in 4 mL of DMF cooled to −10 ° C. was added. When the temperature of the reaction mixture rose to −20 ° C., 3.7 g (15.3 mmol) of 1.05 fold excess of HCl.H-Pro-OBzl was added to 20 ml of DMF and 2.14 ml of TEA. Then, it stirred at 4 degreeC with a magnetic stirrer for 24 hours. The reaction mixture is evaporated, the residue dissolved in ethyl acetate 200 mL, 2 times with _{H 2} O in 20 ml, 3 times with a 10% sodium KHSO ₄ of 20 ml, 3 times with _{H 2} O in 20 ml, 5% of 20 ml NaHCO Washed 3 times with ₃ and 3 times with 3 ml of H ₂ O. The ethyl acetate solution was dried by MgSO ₄ and. It was then evaporated and a small amount of ether (-10 mL) was added to the residue. Then it was left in the refrigerator. A seeding agent was added for better product crystallization. The precipitated crystals were filtered off and washed with a small amount of ether using a filter. It was then dried in a desiccator.
Yield: 5.358 g (11.65 mmol) 79.92%
Rf 0.326 (3); 0.947 (4); 0.390 (5)
Melting point 125-126 ° C
[Α] _D ²² = -101.2 ° (c = 0.85; CH ₃ OH)
Elemental analysis: C62.89 (62.73); N9.21 (9.14); H7.52 (7.24)

IV. TFA H-Pro-Gly-Pro-OBzl. 5.358 g (11.65 mmol) of Boc-Pro-Gly-Pro-OBzl is dissolved in 29.13 ml of methylene chloride and 29.13 mL of TFA is added It was incubated at room temperature for 45 minutes, evaporated twice with absolute ethanol, twice with benzene, twice with ether and dissolved in benzene. Then hexane was poured on. Hexane was decanted and the resulting material was dried in a desiccator under vacuum with P ₂ O ₅ / KOH and paraffin.
Yield: 4.63 g (9.7 mmol) 98%
Rf 0.043 (3); 0.247 (4); 0.018 (5)

_{V. Boc-Arg (NO 2)} -Pro-Gly-Pro-OBzl. Boc-Arg of 3.09 g (9.7 mmol) of (NO ₂₎ was dissolved in THF and 10 ml DMF in 50 ml, then, 2.07 g (10.67 mmol) of DCC was added, cooled to 0 ° C. and stirred for 40 minutes. A solution of TFA.H-Pro-Gly-Pro-OBzl was added to 50 ml of THF and 4.46 ml (9.7 mmol) of TEA. The reaction mixture was stirred for 3 days. The DCM precipitate was filtered off and the solution was evaporated under vacuum, after which 200 ml of hexane was added to the residue. This is used to separate the desired product as an oil, which is dissolved in 500 mL of ethyl acetate, 3 times with 25 ml 0.1 N HCl, 3 times with ml H ₂ O and 1 with a saturated solution of NaCl. Washed twice. The organic layer was dried over MgSO ₄ , filtered and evaporated. The residue was dried under vacuum with P ₂ O ₅ / KOH and paraffin.
Yield: 4.76 g (7.9 mmol) 85%
Rf 0.44 (1); 0.8 (6)
Melting point 108-110 ° C

VI. The _{TFA-Arg (NO 2) -Pro} -Gly-Pro-OBzl. Boc-Arg (NO 2) of 4.76g (7.9mmol) -Pro-Gly- Pro-OBzl was dissolved in 20ml of methylene chloride 20 mL of TFA was added, incubated at room temperature for 45 minutes, evaporated twice with absolute ethanol, twice with benzene, twice with ether and then dissolved in benzene. Hexane was then poured on top. Hexane was decanted and the resulting material was dried in a desiccator under vacuum with P ₂ O ₅ / KOH and paraffin.
Yield is quantitative.
Rf 0.16 (1); 0.27 (6)

VII. Boc-Pro-Arg ( NO 2) -Pro-Gly-Pro-OBzl. Was dissolved Boc-Pro of 1.7 g (7.9 mmol) in THF of 20 mL, BT of 1.07 g (7.9 mmol) Was cooled to 0 ° C. and 1.8 g DCC in 50 ml THF was added. Within 40 minutes, a solution of TFA-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl (7.9 mmol) in 50 mL THF and 1.1 mL (7
. 9 mmol) of TEA was added to the reaction mixture. The mixture was stirred at 0 ° C. for 2 hours and further at room temperature for 2 days. The DCM was then filtered off, evaporated in vacuo, dissolved in 500 mL of ethyl acetate and treated in the same way as Boc-Pro-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl.
Yield: 4.07 g (67.8%)
Rf 0.42 (1); 0.72 (6); 0.31 (7)
Melting point: 147-148 ° C

VIII. Boc-Pro-Arg-Pro-Gly-Pro. 4.07 g (6.5 mmol) is dissolved in 100 ml of methanol and 1 ml of 1 N hydrochloric acid and 0.85 g of catalyst, ie 10 on neutral aluminum oxide % Palladium oxide was added, and hydrogenation was performed for 6 hours while flowing dry hydrogen at room temperature under 1 atm. The catalyst was then filtered off and washed on the filter with methanol. The pooled filtrate was evaporated to dryness. The residue was precipitated from anhydrous methanol with ether.
Yield: 3.02 g (5.8 mmol) 89%
Rf 0.125 (1), 0.57 (2), 0.37 (6)

IX. Pro-Arg-Pro-Gly-Pro. 3.02 g (5.8 mmol) of Boc-Pro-Arg-Pro-Gly-Pro were suspended in 10 ml of 2 N hydrochloric acid in dioxane and incubated for 45 minutes at room temperature .
Thereafter, dry ether was added, and the precipitate was washed by tidal lagoon with dry ether. It was reprecipitated from anhydrous methanol with ether. The resulting precipitate was dissolved in 30% ethanol 7.5ml, Amberlyst A-21 for the acetic acid / hydrochloride salt exchange ^- is applied to (AcO form). The peptide was eluted with 200 ml of 30% ethanol, evaporated to dryness under vacuum and precipitated from methanol with anhydrous ether.
Yield: 2.27 g (75%)
Rf 0.2 (2); 0.1 (6)
Melting point 180-185 ° C
[Α] _D ²⁰ = −105 ° (c = 0.4; CH ₃ COOH)
Amino acid composition versus arginine: Pro 2.78 (3); Gly 1.1 (1)
HPLC result: Column: Supercosil ABZ Plus, size 4.6 × 250 mm, flow rate
1 mL / min, eluent A: NH ₄ H ₂ PO ₄ + H ₃ PO ₄ (50 mM, pH 2.8), eluent B: MeOH
Gradient: 0 to 20 minutes (0 to 40% B), retention time 10.13 minutes

Synthesis of Pro-Gly-Pro tripeptide
The synthesis of Pro-Gly-Pro tripeptide was performed according to the diagram shown in FIG. The synthesis of Pro-Gly-Pro tripeptide was performed using modern protecting groups and methods of peptide bond formation in solution. A mixed anhydride method with PivCl was used for peptide bond formation. Tert-butyloxycarbonyl protection (Boc) was used for protection of the amino group and benzyl ester (OBzl) was supplemented for protection of the carboxyl group. A stepwise approach was used for peptide chain elongation.

  L amino acid derivatives were used in the synthesis. Evaporation of the solution was carried out at 40 ° C. using a vacuum evaporator. The melting point determined on the Boethius device is shown without calibration.

The identity of the compound obtained was determined on TL on Silufol silica gel coated plates (Czech Republic).
Tested by C. Substances were detected by spraying a solution of ninhydrin and / or o-tolidine onto the plate. Chromatographic transfer rate (Rf) values in the following solvent systems are shown. (Ethyl acetate: acetone: 50% acetic acid: water (2: 1: 1); benzene: ethanol (8: 2); chloroform: methanol: ammonia (6: 4: 1); chloroform: methanol: acetic acid (42: 7 Acetone: benzene: acetic acid (50: 100: 1); chloroform: methanol (9: 1); hexane: acetone (3: 2); butanol: acetic acid: water (4: 1: 1); butanol: Acetic acid: pyridine: water (30: 6: 20: 24); hexane: ethyl acetate (4: 1); chloroform: methanol: ammonia (8: 1.75: 0.25); (isopropanol: formic acid: water) (20: 5: 1); (chloroform: methanol: ammonia) (7: 2.5: 0.5); methanol. Specific rotation was determined by AI-EPO polarimeter. It was carried out using a Carlo-Erba model 1106 analyzer.

I. Production of Boc-Pro-Gly-OH 10.75 g (50 mmol) of Boc-Pro was dissolved in 150 ml of acetonitrile and cooled to −5 ° C., after which 7.7 ml (50 mmol) of triethylamine (TEA) was added to the solution. It cooled to -20 degreeC, stirring with a magnetic stirrer. 6.
Add 8 ml (55 mmol) of pivaloyl chloride (PivCl) to the cooled solution and stir with a magnetic stirrer at -10 ° C for 20 minutes, then cool to -30 ° C and then pre-chilled solution of Gly Was added. At the same time, a Gly solution was prepared.

2. 4.5 g Gly (60 mmol, 1.2-fold excess) was dissolved in 35 ml water and 60 ml acetonitrile and 8.4 ml (60 mmol) triethylamine was added. The mixture was cooled to −10 ° C. and added to the solution in the first flask after 20 minutes. The reaction mixture was incubated at −10 ° C. for 1 hour and stirred with a magnetic stirrer at 18-20 ° C. for 2 hours. The reaction mixture was evaporated on a rotary evaporator. Approximately 50 mL of water was added to the residue. The aqueous solution was acidified to pH = 3 with a 3-fold excess of NaHSO ₄ (24.84 g) and extracted 5-fold with 100 ml of ethyl acetate. The pooled ethyl acetate solution _{H 2 O (50mL), 10} % solution of KHSO 4 _(50mL), washed with H 2 O (50mL), and saturated NaCl (50 mL). The ethyl acetate solution was dried by MgSO ₄ and. The dried ethyl acetate was filtered and evaporated. Dry ether was added to the residue. When ether was added to the flask, the product precipitated and was filtered and washed with dry ether using a filter. The resulting material was dried under vacuum with KOH, P ₂ O ₅ and paraffin in a desiccator while changing the desiccator several times.
Product M.I. W. 272.3
Yield: 5.97 g (21.74 mmol); (43.5%)
Melting point 70 ° C
Rf 0.863 (acetone-benzene-acetic acid) (50: 100: 1);
0.746 (benzene-ethanol) (8: 2); 0.903 (chloroform: methanol) (9: 1);
0.847 (ethyl acetate: acetone: 50% acetic acid: water) (2: 1: 1)

II. Production of Boc-Pro-Gly-Pro-OBzl 5.97 g (21.74 mmol) of Boc-Pro-Gly-OH were dissolved in 100 ml of acetonitrile and cooled to -5.degree. Thereafter, a 1.1-fold excess (3.35 ml, 23.9 mmol) of triethylamine (TEA) was added to the solution and cooled to −20 ° C. with magnetic stirring. Add a 1.1-fold excess (2.34 ml, 23.9 mmol) of pivaloyl chloride (PivCl) to the cooled solution and stir with a magnetic stirrer at -10 ° C for 20 minutes, then cool to -30 ° C. Thereafter, a pre-cooled solution of HCl.Pro-OBzl was added. At the same time, HCl.Pro-OBzl was prepared.

2. 6.3 g of HCl Pro-OBzl (26.1 mmol, 1.2 fold excess) was dissolved in 50 ml of acetonitrile and 4.0 ml (28.71 mmol, 1.1 fold excess) of triethylamine was added. The mixture was cooled to −10 ° C. and added to the solution in the first flask after 20 minutes. The reaction mixture was incubated at −10 ° C. for 1 hour and stirred with a magnetic stirrer at 18-20 ° C. for 2 hours. The reaction mixture was evaporated. To the evaporated residue was added 300 ml of ethyl acetate. Ethyl acetate solution: H ₂ O (3 × 25 mL), 10% solution KHSO ₄ (3 × 25 ml), H ₂ O (3 × 25 ml), 5% NaHCO ₃ (3 × 25 ml), H ₂ O (3 × 25 mL) and washed with a saturated solution of NaCl (1 × 25 mL). The ethyl acetate solution was dried by MgSO ₄ and. The dried ethyl acetate was filtered and evaporated. About 100 μm of dry ether was added to the residue. When ether was added to the flask, the product precipitated and was filtered and washed with dry ether using a filter. The resulting material was dried under vacuum in a desiccator with KOH, P ₂ O ₅ and paraffin while changing the desiccant several times.
Product M.I. W. 459.82
Yield: 8.12 g (17.66 mmol); (81.23%)
Melting point 125-126 ° C
Rf 0.326 (acetone: benzene: acetic acid) (50: 100: 1)
0.390 (hexane: acetone) (3: 2)
0.947 (chloroform: methanol) (9: 1)
0.716 (methanol)
0.620 (benzene: ethanol) (8: 2)

III. Production of Boc-Pro-Gly-Pro-OH 8 g (17.4 mmol) of Boc-Pro-Gly-Pro-OBzl was dissolved in 100 ml of methanol and stirred with a magnetic stirrer while adding 0.5 ml of CH. ₃ COOH and palladium black were added and hydrogen was flowed for 2 hours. The solution was filtered and evaporated, evaporated three times with benzene and twice with ethyl acetate. Thereafter, precipitated with ether hexane from acetone, the precipitate formed in the flask was dried in a desiccator with P _{2 O} 5 / _KOH and paraffin.
Product M.I. W. 369.39
Yield: 6.3 g (17.05 mmol) 98%
Melting point 99-100 ° C
Rf 0.560 (chloroform-methanol) (9: 1)
0.812 (chloroform-methanol-acetic acid) (42: 7: 1)
0.187 (acetone: benzene: acetic acid) (50: 100: 1)
0.164 (hexane: acetone) (3: 2)

IV. Production of Pro-Gly-Pro 40.6 ml of methylene chloride and 40.6 ml of TFA were added to 6.0 g (16.24 mmol) of Boc-Pro-Gly-Pro-OH and incubated for 45 minutes at room temperature. . After removal of the protecting group, the solution was evaporated twice with anhydrous methanol, twice with benzene and twice with ether. Precipitated from acetone with ether. The residue was dried under vacuum with P ₂ O ₅ , KOH and paraffin. The dried product was reprecipitated from anhydrous MeOH with dry diethyl ether.
Product M.I. W. 381.39
Yield: 5.6 g (14.72 mmol) (90.62%)

The resulting precipitate of TFA-Pro-Gly-Pro-OH is dissolved in 10 ml of 30% ethanol and then 25 ml of Amberlyst A-21 (CH ₃ COO) for the exchange of trifluoroacetate to acetate. ^- ) Ion-exchange resin was added and stirred for 45 minutes with a magnetic stirrer. Thereafter, it was washed with 150 ml of 30% ethanol, evaporated to dryness under vacuum, and precipitated from anhydrous methanol with dry diethyl ether. The resulting precipitate was filtered off and dried in a desiccator under vacuum with P ₂ O ₅ , KOH and paraffin, while the desiccant was changed several times.
Product M.I. W. 328.34
Yield: 4.54 g (13.84 mmol) (94%)
Melting point: 143-145 ° C
[Α] _D ²² -31.5 ° (cl, MeOH)
Rf 0.59 (chloroform: methanol: ammonia) (6: 4: 1)
0.52 (chloroform: methanol: ammonia) (4: 4.5: 1.5)
0.524 (ethanol: ammonia) (7: 3)

Synthesis of Thr-Lys-Pro-Arg-Pro-Phe hexapeptide
The synthesis of Thr-Lys-Pro-Arg-Pro-Phe hexapeptide was performed according to the diagram shown in FIG.

The synthesis of Thr-Lys-Pro-Arg-Pro-Phe hexapeptide was performed using modern protecting groups and methods of peptide bond formation in solution. A TBA salt method, an activated ester method, a carbodiimide method and a mixed anhydride method were used for peptide bond formation. Both staged and block approaches were used.

Synthesis of Thr-Lys-Pro-Arg-Pro pentapeptide
Thr-Lys-Pro-Arg-Pro pentapeptide was synthesized according to the diagram shown in FIG.

The synthesis of Thr-Lys-Pro-Arg-Pro pentapeptide was performed using modern protecting groups and methods of peptide bond formation in solution. TBA salt method, activated ester method and carbodiimide method were used for peptide bond formation. Both staged and block approaches were used.

Synthesis of Thr-Lys dipeptide
The synthesis of Thr-Lys dipeptide was performed according to the diagram shown in FIG.

The synthesis of Thr-Lys dipeptide was performed using modern protecting groups and methods of peptide bond formation in solution. The TBA salt method was used for peptide bond formation.

  For the synthesis of the peptides described in Examples 7-9, both protected and free L amino acid derivatives were used. The solvent used for peptide synthesis was correspondingly dehydrated. Peptide homogeneity was tested by high performance liquid chromatography (HPLC) using a Milichrome-A02 microcolumn liquid chromatography system. The synthesized peptides were characterized by mass spectrometry using a Bruker Mass Spectrometer (® Bruker Daltonics).

Synthesis of Thr-Lys-Pro-Phe tetrapeptide
The Thr-Lys-Pro-Phe tetrapeptide was synthesized according to the diagram shown in FIG.

The synthesis of the Thr-Lys-Pro-Phe tetrapeptide was carried out using modern protecting groups and methods of peptide bond formation in solution. The TBA salt method and the activated ester method were used for peptide bond formation. A stepwise approach to peptide chain elongation was used.

Both protected and free derivatives of L-amino acids were used for the synthesis. The solution was evaporated using a vacuum evaporator at 40 ° C. The melting point determined on the Boethius device is shown without calibration. The identity of the resulting compounds was tested by TLC on Silufol silica gel coated plates (Czech Republic). Substances were detected by spraying a solution of ninhydrin and / or o-tolidine onto the plate. Chromatographic transfer rate (Rf) values in the following solvent systems are shown. (Butanol: acetic acid: water) (4: 1: 1); (benzene-ethanol) (8: 2); (chloroform: methanol) (9: 1); (isopropanol: formic acid: water) (20: 5: 1); (Chloroform-methanol-acetic acid) (42: 7: 1); (Chloroform: methanol: ammonia) (8: 1. 75: 0.25); (Acetone-benzene-acetic acid) (50: 100: 1) (Chloroform: methanol: ammonia) (6: 4: 1); (chloroform: methanol: ammonia) (44.5: 1.5); (butanol: acetic acid: pyridine: water) (30: 6: 20) 24).

I. Production of Z-Lys (Boc) -Pro-OH A 13% solution of TBA (98 ml) was added to 46.32 mmol (5.34 g) of Pro, twice with ethanol, twice with an ethanol / benzene mixture, with benzene. Evaporated twice. 300 ml of anhydrous ethyl acetate was added. The reaction mixture was cooled to 0 ° C., 23.16 mmol (12.66 g) of the previously synthesized Z-Lys (Boc) -OPfp was added, and the mixture was stirred for 1 hour with a magnetic stirrer. The reaction mass was evaporated and 40 ml water was added to the evaporation residue. The aqueous solution was washed three times with 80 ml of ether. After washing with ether, the aqueous solution was acidified to pH 3 with citric acid. After acidification, the aqueous solution was extracted three times with 40 ml aliquots of ethyl acetate. After extraction, the pooled ethyl acetate was washed three times with 20 ml of water, three times with 20 ml of a 10% solution of KHSO ₄ and three times with 20 ml of water. The ethyl acetate solution was dried by MgSO ₄ and. It was then filtered and evaporated on a rotary evaporator. The resulting material was precipitated from ethyl acetate with hexane. Hexane was decanted and the resulting material was dried under vacuum with KOH, P ₂ O ₅ and paraffin in a desiccator, while the desiccant was repeatedly exchanged.
Yield: 9.68 g (20.26 mmol); (87.51%)
Melting point 76-77 ° C
Rf 0.705 (butanol: acetic acid: water) (4: 1: 1);
0.560 (benzene-ethanol) (8: 2);
0.476 (chloroform: methanol) (9: 1);
0.813 (isopropanol: formic acid: water) (20: 5: 1);
0.297 (acetone-benzene-acetic acid) (50: 100: 1)

I. Preparation of H-Lys (Boc) -Pro-OH To 9.68 g (20.26 mmol) of Z-Lys (Boc) -Pro-OH was added 300 ml of anhydrous methanol, 2 ml of acetic acid, and palladium black, under 1 atm. Hydrogenation was carried out for 8 hours while flowing dry hydrogen at room temperature. The catalyst was then filtered off and washed on the filter with methanol. The pooled filtrate was evaporated to dryness. The residue was precipitated from anhydrous methanol with ether. Then, it was made to dry under vacuum and during that time, the desiccant was changed several times.
Yield: 6.38 g (18.58 mmol); (91.87%)
Melting point 98-99 ° C
Rf 0.110 (chloroform-methanol-acetic acid) (42: 7: 1);
0.166 (butanol: acetic acid: water) (4: 1: 1);
0.235 (chloroform: methanol: ammonia) (8: 1.75: 0.25);
0.494 (isopropanol: formic acid: water) (20: 5: 1) (2: 1: 1)

III. Preparation of Boc-Thr- Lys- (Boc) -Pro-OH A 13% solution of TBA (39.4 ml) is added to 18.58 mmol (6.38 g) of Lys (Boc) -Pro-OH and ethanol 2 Evaporated twice with ethanol / benzene mixture and twice with benzene. 250 ml of anhydrous ethyl acetate was added, the reaction mixture was cooled to 0 ° C., 9.2 mmol (3.74 g) of the previously synthesized Boc-Thr-OPfp was added and stirred with a magnetic stirrer for 1 hour. The reaction mass was evaporated and 40 ml water was added to the evaporation residue. The aqueous solution was washed three times with 80 ml of ether. After washing with ether, the aqueous solution was acidified to pH 3 with 18.58 mmol (3.95 g) citric acid. After acidification, the aqueous solution was extracted three times with 40 ml aliquots of ethyl acetate. After extraction, the pooled ethyl acetate was washed three times with 20 ml of water, three times with 20 ml of a 10% solution of KHSO ₄ and three times with 20 ml of water. The ethyl acetate solution was dried by MgSO ₄ and.

It was then filtered and evaporated on a rotary evaporator. The resulting material was precipitated from ethyl acetate with hexane. Hexane was decanted and the resulting material was dried under vacuum in a desiccator with KOH, P ₂ O ₅ and paraffin while the desiccant was changed several times.
Yield: 3.32 g (6.1 mmol); (66.26%)
Melting point 105-107 ° C
Rf 0.297 (acetone-benzene-acetic acid) (50: 100: 1);
0.234 (chloroform: methanol) (9: 1);
0.560 (benzene-ethanol) (8: 2)

IV. Production of Boc-Thr-Lys (Boc) -Pro-Phe-OH Production of Boc-Thr-Lys (Boc) -Pro-OSu 3.53 mmol (0.38 g) of hydroxysuccinimide in 1.66 g (3.05 mmol) of Boc-Thr-Lys (Boc) in 50 ml of anhydrous ethyl acetate The resulting solution was cooled to 0 ° C. while adding to -Pro and stirring with a magnetic stirrer. Then, 0.76 g (3.53 mmol) DCC (dicyclohexyl carbodiimide) was added, and it stirred by magnetic stirrer for 2 hours at room temperature. After termination of the reaction, the resulting reaction mixture was filtered off and the precipitate was discarded. To the resulting solution was added 200 ml of anhydrous ethyl acetate. Twice pooled ethyl acetate with a saturated solution of NaCl in 20ml, 2 times with 10% sodium KHSO ₄ of 20ml, 2 times with a saturated solution of NaCl in 20ml, 2 times with 5% MaHCO ₃ of 20ml, NaCl in 20ml Washed twice with a saturated solution of. The ethyl acetate solution was dried by MgSO ₄ and. It was then filtered and evaporated on a rotary evaporator. The resulting material was precipitated from ethyl acetate with ether and hexane. The precipitate was filtered and dried under vacuum with KOH, P ₂ O ₅ and paraffin, while changing the desiccant several times.
Yield: 1.52 g (2.37 mmol) 77.74%
Rf 0.560 (benzene-ethanol) (8: 2); 0.457 (chloroform: methanol) (9: 1)

2. After the preparation, 1.52 g (2.37 mmol) of Boc-Thr-Lys (Boc) -Pro-OSu was added to 25
Dissolved in ml dimethylformamide and added to the prepared solution containing 0.392 g (2.37 mmol) L-Phe in 25 ml dimethylformamide. The solution was stirred with a magnetic stirrer at room temperature. The reaction mixture was evaporated in a rotary evaporator and precipitated from benzene with ether. The precipitate was filtered off and dried under vacuum with P ₂ O ₅ / KOH and paraffin, while changing the desiccant several times.
Yield: 1.03 g (1.64 mmol) 69.0%
Rf 0.063 (isopropanol: formic acid: water) (20: 5: 1) (2: 1: 1)
0.745 (chloroform: methanol: ammonia) (8: 1.75: 0.25)

V. Production of H-Thr-Lys-Pro-Phe-OH 1.03 g (1.64 mmol) of Boc-Thr-Lys (Boc) -Pro-Phe-OH into 8.2 mL of methylene chloride and 8.2 ml of TFA After that, the mixture was incubated at room temperature for 45 minutes and after removal of the protecting group, the solution was evaporated twice with anhydrous methanol, twice with benzene and twice with ether. Precipitation from methanol with ether. The residue was dried under vacuum with P ₂ O ₅ KOH and paraffin. The resulting precipitate was dissolved in 30% ethanol 5ml, Amberlyst A-21 for the acetic acid / hydrochloride salt exchange (AcO ^- form) was applied to the column. The peptide was eluted with 100 ml 30% ethanol, evaporated to dryness under vacuum and precipitated from anhydrous methanol with anhydrous ether. The resulting precipitate was filtered and dried in a desiccator under vacuum with P ₂ O ₅ , KOH and paraffin, while the desiccant was changed several times.
Yield: 0.7 g (1.43 mmol) (87%)
Melting point: 129-131 ° C
Rf 0.201 (butanol: acetic acid: pyridine: water) (30: 6: 20: 24);
0.156 (chloroform: methanol: ammonia) (4: 4.5: 1.5)

  The chromatographic and mass spectrometric analysis of the peptide sequences described in Examples 1-7 is shown in Table 2.

  Table 3 shows the gradient shape for the separation of synthetic peptides.

Identification of Pharmacophore Position In order to identify the pharmacophore, a fragment of the parent peptide Selank, Thr-Lys; Thr-Lys-Pro; Pro-Gly-Pro; Arg-Pro-Gly-Pro; Pro-Arg-Pro -Gly-Pro was synthesized and tested for efficacy in vivo using the relevant preclinical model (Lordosis test).

We determined that, at a dose of 100 μg / rat, Thr-Lys; Thr-Lys-Pro; Pro-Gly-Pro; Arg-Pro-Gly-Pro; Pro-Arg-Pro- at a dose of 100 μg / rat in relation to the female rat's sexual behavior. The effectiveness of the group of peptides consisting of Gly-Pro was studied. Sexual behavior was recorded in ovariectomized, hormone-stimulated females who were in direct contact with sexually active males or when such contact was not possible. During monitoring, it was observed that the Thr-Lys-Pro peptide increased the strength of the proceptive behavior in females from 14 ± 4 to 29 ± 6 (p = 0.0). 028, Wilcoxon test). The effect on lordosis response in females has a similar tendency (p = 0.09), and the number of lordosis under the action of Thr-Lys-Pro peptide is 0.73 ± 0.
. It increased from 12 to 0.97 ± 0.12. These results indicate an increase in sexual motivation behind the Thr-Lys-Pro peptide action. The effects are specific and are expressed under appropriate behavioral conditions. Table 4 shows the results of efficacy studies on peptides of Thr-Lys; Thr-Lys-Pro; Pro-Gly-Pro; Arg-Pro-Gly-Pro; and Pro-Arg-Pro-Gly-Pro in the Rhodes model Show.

Pharmacophore Test To test pharmacophores, Pharmacophore-based peptides, ie, Thr-Lys-Pro tripeptide, Thr-Lys-Pro, corresponding to the general formula A-Thr-Lys-Pro-BCDX. -Arg
And Thr-Lys-Pro-Phe tetrapeptide, Thr-Lys-Pro-Arg-Pro pentapeptide, and Thr-Lys-Pro-Arg-Pro-Phe hexapeptide synthesized and effective using relevant preclinical models Sex tests were performed in vivo (Lodesis test).

We found that, at a dose of 100 μg / rat, Thr-Lys-Pro; Thr-Lys-Pro-Arg; Thr-Lys-Pro-Arg-Pro; and Thr-Lys-Pro in relation to the sexual behavior of female rats. The efficacy of the group of peptides consisting of -Arg-Pro-Phe was studied. Sexual behavior was recorded in ovariectomized and hormonally stimulated females when in direct contact with sexually active males or when such contact was not possible. During monitoring, peptides from the group containing Thr-Lys-Pro, Thr-Lys-Pro-Arg-Pro, and Thr-Lys-Pro-Arg-Pro-Phe have strong mating-favoring behavior in females Was observed to increase from 14 ± 4 to 26 ± 4 to 36 ± 6 (p = 0.028, Wilcoxon test). At the same time, Thr-Lys-Pro-Arg and Thr-Lys-Pro-Phe do not affect the strength of the proceptive behavior in female matings, and do not increase the number of lordosis, The basic parameters of the Lys-Pro-Arg and Thr-Lys-Pro-Phe tetrapeptide remain at the level of the negative control. The effect on the rhodocis reaction in females had a similar tendency (p = 0.09). The peptide action was manifested even without direct contact of the partners. The results show that sexual motivation is enhanced by the peptide action of Thr-Lys-Pro, Thr-Lys-Pro-Arg-Pro, and Thr-Lys-Pro-Arg-Pro-Phe, and Thr-Lys-Pro- FIG. 3 shows the lack of sexual motivation effects in Arg and Thr-Lys-Pro-Phe tetrapeptides. The effects are specific and are expressed under appropriate behavioral conditions. Efficacy of the peptide Thr-Lys-Pro; Thr-Lys-Pro-Arg; Thr-Lys-Pro-Phe; Thr-Lys-Pro-Arg-Pro; Thr-Lys-Pro-Arg-Pro-Phe in a model of Rhodosis The results of the sex study are shown in Table 5.

Industrial applicability
The invention relates to the field of biochemistry and in particular to a method for producing peptides that exhibit very high activity and are capable of stimulating natural healing in damaged organs. In particular, the invention makes it possible to expand the range of techniques for stimulating sexual function and treating sexual dysfunction while reducing the duration of the course of therapy and reducing the cost of the drug.

Claims (11)

A pharmaceutical composition for stimulating sexual or reproductive functions in a mammal in need stimulation of sexual or reproductive function, one general formula:
Thr-Lys-Pro-X
(Wherein X is C-terminal OH, OCH ₃ , NH ₂ )
A pharmaceutical composition comprising a peptide having General formula:
Thr-Lys-Pro-Arg-Pro-X
(Wherein X is C-terminal OH, OCH ₃ , NH ₂ )
A pharmaceutical composition comprising a peptide having General formula:
Thr-Lys-Pro-Arg-Pro-Phe-X
(Wherein X is C-terminal OH, OCH ₃ , NH ₂ )
A pharmaceutical composition comprising a peptide having The pharmaceutical composition according to any one of claims 1 to 3 , wherein the peptide is produced by expression in a recombinant microorganism. The pharmaceutical composition according to any one of claims 1 to 3 , wherein the peptide is produced by chemical synthesis. The pharmaceutical composition according to any one of claims 1 to 4 , wherein the peptide is present in the composition in the form of a salt. Use of the pharmaceutical composition according to any one of claims 1 to 6 for the manufacture of a medicament for stimulating sexual or reproductive function in a mammal in need of stimulation of sexual or reproductive function. 8. Use according to claim 7 , wherein the mammal has reproductive or sexual dysfunction. 7. A medicament for the treatment or prophylaxis of reproductive or sexual dysfunction in a mammal in need of the treatment or prophylactic treatment of reproductive or sexual dysfunction according to any one of claims 1 to 6. Use of a pharmaceutical composition. The pharmaceutical composition according to claim 2, which is for stimulating sexual or reproductive function in a mammal requiring sexual or reproductive stimulation . The pharmaceutical composition according to claim 3, which is for stimulating sexual or reproductive function in a mammal in need of stimulation of sexual or reproductive function .

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