RU2087480C1 - Peptide derivatives and method of their production - Google Patents

Abstract

FIELD: medicine, biology, particular, methods of peptide derivatives production. SUBSTANCE: product - derivatives of peptides of formula l: A-Ile-Y-Phe-B, where A-H, HVal, HSer- X-Val, Glp-Pro-Pro-Gly-Gly-Ser-X-Val; B - OH, Arg-OH; X - Lys, Arg; Y - Leu, Tie. Reagent 1: C-terminal amino acid. Reaction conditions: (1) successive building up of peptide groups with the help of activated esters protected by alpha-amino group of amino acids to obtain compounds of formula 1, where A -H, HVal, HSer-X-Val, which are subjected to fragmentary condensation in the presence of decyclohexylcarbonylimide and precluding racenization of additive with N-terminal compound: Glp-Pro-Pro-Gly-Gly-Ser-X-Val; (2) production of peptide derivatives of the formula: A-Ile-Y-Phe-OH, where A - Glp-Pro-Pro-Gly-Gly-Ser-X-Val is carried out by successive building up of group, beginning from C-terminal fragment up to obtaining N-terminal pentapeptide: Glp-Pro-Pro-Gly-Gly, dipeptide Ser-Arg and C- terminal tetrapeptide: Val-Ile-Leu-Phe, with condition of condensation of obtained fragments by successive attachment to above-mentioned N-terminal pentapeptide of dipeptide Set-Arg with the help of decyclohexylcarbonylimide in the presence of additive precluding racemization and attachment to obtained hexapeptide Glp-Pro-Pro-Gly-Gly-Ser-Arg of C-terminal tetrapeptide Val-Ile-Leu-Phe with the help of diphenylphosphoryazide. EFFECT: higher efficiency. 13 tbl

Description

 The invention relates to bioorganic chemistry, more specifically the chemistry of peptides, and even more specifically the invention relates to biologically active structural analogues of a natural peptide isolated from Hydra attenuata and a method for producing such analogues.

 The invention may find application in the creation of new highly effective drugs.

 It has now been established that more than a dozen peptides identical to the neuropeptides of the human brain are represented in the brain tissue of primitive chordates. An active study of such peptides is of practical importance, since the identification of the specific functions of phylogenetically ancient peptides in higher animals can serve as the basis for the creation of new highly effective drugs.

One of the known phylogenetically ancient peptides is a natural peptide isolated from Hydra attenuata. This substance is a undecapeptide with a molecular weight of 1124 Da and an amino acid sequence
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe-OH.

 In Hydra attenuata, the peptide is a factor in the growth and differentiation of tissues and activates the regeneration of the head end of her body.

 A peptide of identical amino acid sequence has been found in the blood plasma of mammals, including humans; It is believed that this peptide can act as a growth factor in the normal development of higher animals and humans, and can also regulate body functions in various pathological conditions.

 To conduct studies to clarify the biological functions of the named undecapetide isolated from Hydra attenuata, significant quantities of this substance are required, which can be obtained only by chemical synthesis.Natural analogues of this peptide have not been identified, and all synthesized analogues have no biological activity characteristic of a natural peptide.

 The following peptides [1] (1) Clp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe-OH (2) H-Tyr-Cln-Pro-Pro-Cly were obtained by chemical synthesis -Gly-Ser-Lys-Val-Ile-Leu-Phe-OH (3) Glp-Pro-Pro-Gly-Gly-Ser-Lys-Pro-Val-Ile-Leu-Phe-OH.

 The synthesis was carried out by the solid phase method, a, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl group was used as a protective group for the α-amino function, a benzyloxycarbonyl protection was used to block the lysine e-amino group, a tert-butyl group was used to protect the aromatic and tyrosine hydroxyl group, 4, The 4-dimethoxybenzylhydryl protecting group was used in the case of glutamine.

After removal from the resin using HBr in trifluoroacetic acid and subsequent purification on Sephodex LH-20, the following peptides were obtained: (1) with a yield of 18% (2) with a yield of 47% and (3) with a yield of 33%
The resulting compounds were eluted at one peak under high performance liquid chromatography (HPLC) with the release time of the peptide (1) coinciding with the release time of the native peptide. Joint chromatography of these two peptides, synthetic and native, also gave one peak.

 Of the three synthesized peptides, only peptide (1) exhibits biological activity identical to the natural growth activator.

 Removing one amino acid from sequence (1), for example, glycine or proline, replacing the N-terminal residue of pyroglutamic acid with glutamic acid or glutamine, introducing one additional tyrosine amino acid at the N-terminus or proline between lysine and valine, all this leads to a loss of biological activity the resulting peptide. By-products present in the reaction mixture, which are peptides with the amino acid sequence of compound (1), but with the replacement of a number of L-amino acids by their D-isomers, do not possess biological activity.

The basis of the claimed invention is the task of creating new substances
structural analogues of a natural peptide isolated from Hydra attenuata, which have biological activity that allows them to be used both as synthetic stimulators of regeneration processes in mammalian organs and tissues, and as inhibitors of pathological growth processes in mammalian tissues, as well as a method for producing such structural analogues.

 This problem is solved in that the biologically active structural analogue of a natural peptide isolated from Hydra attenuata according to the invention is a peptide of the following amino acid sequence: A-Ile-Y-Phe-B, where A: H-, H-Val-, H- Ser-X-; Glp-Pro-Pro-Gly-Gly-Ser-X-Val-; B: -OH, -Arg-OH; X: -Lys-, -Arg-; Y: -Leu-, -Tle-, due to which it exhibits the properties of a regulator of the processes of regeneration and growth of tissues, and a regulator of the endocrine system.

An embodiment of the claimed invention is that the biologically active structural analogue of a natural peptide isolated from Hydra attenuata is a undecapeptide of the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH,
which exhibits the properties of a synthetic stimulator of regeneration processes in organs and tissues.

Another variant of the claimed invention is that the biologically active structural analogue of a natural peptide isolated from Hydra attenuata is a undecapeptide of the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Tle-Phe-OH,
which exhibits the properties of an inhibitor of growth processes in mammalian tissues.

Another variant of the claimed invention is that the biologically active structural analogue of a natural peptide isolated from Hydra attenuata is a hexapeptide of the following amino acid sequence:
H-Ser-X-Val-Ile-Leu-Phe-OH, X: -Lys-, -Arg-,
which exhibits the properties of a stimulator of regeneration processes.

An embodiment of the claimed invention consists in the fact that a biologically active structural analogue of a natural peptide isolated from Hydra attenuata is a tetrapeptide of the following amino acid sequence:
H-Val-Ile-Y-Phe-OH, Y: -Leu-,
which exhibits the properties of a stimulator of regeneration processes.

An embodiment of the claimed invention consists in the fact that a biologically active structural analogue of a natural peptide isolated from Hydra attenuata is a peptide of the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Lle-Phe-Arg-OH
or the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Lle-Phe-Arg-OH,
which have the property of stimulating the processes of regeneration in organs and tissues.

In accordance with the claimed invention, it is advisable to biologically active structural analogues of a natural peptide isolated from Hydra attenuata, to obtain a method involving the sequential extension of the peptide chain from the C-end of the amino acid sequence using an activated ester of an amino acid protected by an alpha-amino group in which, according to the invention, sequential peptide chain extension is carried out to obtain a peptide:
A-ile-y-phe-b
where A: H-, H-Val, H-Ser-X;
B: -OH, -Arg-OH;
X: -Lys-, -Arg-;
Y: -Leu, -Tle-,
then carry out fragment condensation using dicyclohexylcarbodiimide in the presence of an additive that excludes racemization, to obtain a peptide of the following sequence:
A-Ile-Y-Phe-B,
where A: Glp-Pro-Pro-Gly-Gly-Ser-X-Val-,
B, X and Y have the above meanings.

In accordance with the claimed invention, it is advisable when obtaining the peptide of the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH or
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Tle-Phe-OH or
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Lle-Phe-Arg-OH or
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Lle-Phe-Arg-OH,
use a method in which the sequential extension of the peptide chain is carried out to obtain the N-terminal pentapeptide of the following amino acid sequence: Glp-Pro-Pro-Gly-Gly- and C-terminal hexapeptide with the following amino acid sequence:
Ser-X-Val-Ile-Y-Phe-B,
X: -Lys-, -Arg-; Y: -Leu-, -Tle-; B: -OH, -Arg-OH.

In accordance with the claimed invention, it is advisable when obtaining the peptide of the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH
use a method in which sequential peptide chain extension is carried out to obtain the N-terminal pentapeptide of the following Glp-Pro-Pro-Gly-Gly amino acid sequence, Ser-Arg dipeptide and Val-Ile-Leu-Phe C-terminal tetrapeptide, and condensation of the obtained peptide fragments are carried out by sequentially attaching the Ser-Arg dipeptide to the N-terminal pentapeptide with dicyclohexylcarbodiimide in the presence of an anti-racification additive and adding Glp-Pro-Pro-Gly-Gly-Se to the obtained N-terminal heptapeptide r-Arg of the C-terminal Val-Ile-Leu-Phe tetrapeptide using diphenylphosphorylazide.

 Further objectives and advantages of the claimed invention will become apparent from the following detailed description of biologically active structural analogues of a natural peptide isolated from Hydra attenuata, a method for their preparation, examples of specific claimed compounds and experimental testing of the claimed compounds.

In accordance with the claimed invention, a biologically active structural analogue of a natural peptide isolated from Hydra attenuata is provided, which is a peptide of the following amino acid sequence:
A-Ile-Y-Phe-B,
where A: H-, H-Val-; H-Ser-X; Glp-Pro-Pro-Gly-Gly-Ser-X-Val;
X: -Lys-, -Arg-;
Y: -Leu-, -Tle-;
B: -OH, -Arg-OH.

The structure of the claimed compounds is confirmed by amino acid analysis, ¹ H-NMR spectroscopy and mass spectroscopy.

The resulting compounds are characterized by the specific rotation [α] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ melting point, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).

 The inventive peptide compounds are obtained by classical methods of peptide chemistry in solution using, if necessary, fragment condensation.

So, for example, upon receipt of a undecapeptide with the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH
or undecapeptide with the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Tle-Phe-OH,
or a peptide with the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Lle-Phe-Arg-OH
or peptide with the following amino acid sequence
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Lle-Phe-Arg-OH,
carry out sequential peptide chain extension from the C-end of the amino acid sequence of the fragment to obtain the N-terminal pentapeptide of the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly
and a C-terminal peptide with the following amino acid sequence:
Ser-X-Val-Ile-Y-Phe-B,
where X: -Lys-; -Arg-;
Y: -Leu-, -Tle-;
B: -OH; -Arg-OH.

 The growth of the peptide chain is carried out using an activated ester of an amino acid protected by an alpha amino group, for example, n-nitrophenyl ether, pentachlorophenyl ether, N-hydroxy-5-norbornene-2,3-dicarboxyimide ester of carbobenzoxyamino acid.

 The next step in the preparation of these undecapeptides is the condensation of fragments obtained by sequentially building up the peptide chain to form the desired product, the condensation being carried out using dicyclohexylcarbodimide in the presence of an additive that excludes racimization, for example, in the presence of N-hydroxy-5-norbonen-2,3-dicarboxiimide .

 When the peptide chain is sequentially built up in accordance with the indicated procedure, the excess of activated ester and n-nitrophenol or HOCp or HOHB released as a result of the reaction are removed using a solvent in which the main reaction product is insoluble, i.e., reprecipitation. In some cases, when the solubility of the target product and p-nitrophenol was close, for example, C-terminal di- and tri-peptides, p-nitrophenol is removed by treatment with amberite IRA-410 in the OH form, controlling the process using TLC.

The choice of a combination of protective groups largely determines the conditions for further synthesis. For example, the use of N ^α -protecting groups of the urethane type regardless of the condensation method reduces the likelihood of racemization. It was found that when the peptide chain is stepped up from the C-terminus by the method of activated esters and the use of urethane-type protections is used, condensation is not accompanied by racemization.

 The choice of a combination of protective groups depends, in turn, on a specific amino acid sequence and is determined by the possibility of selective release of temporary and permanent protective groups. Since the structural analogues of the natural peptide isolated from Hydra attenuata lack an amino acid sulfur content, the benzyloxycarbonyl group that is cleaved by hydrogenolysis is selected as a temporary protective group, while the tert-butyl groups that are resistant to hydrogenolysis and are cleaved by acidolysis are used as permanent protective groups. This combination of protective groups is used in the synthesis of fragments according to the above procedure: a benzyloxycarbonyl protective group (Z) is used as a temporary protection of the α-amino group, a tert-butyloxycarbonyl protective group (Boc) is used to permanently block the e-amino group of lysine, phenylalnine is reacted in the form of tert-butyl ether, a tert-butyl group is used to protect the hydroxyl function of serine.

The use of the activated esters method in stepwise synthesis allows for minimal protection of functional groups. Thus, the carboxyl group of the C-terminal glycine in the synthesis of the Glp-Pro-Pro-Gly-Gly fragment is protected by salt formation, the serine hydroxyl group in the synthesis of peptides
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Tle-Phe-OH and
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe-Arg-OH
leave unprotected, and in other cases, tert-butyl protection is used.

When receiving peptides with Arg in position 7, that is
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-Arg-OH
at the stage of arginine addition, t-carboxybenzoxy arginine p-nitrophenyl ether is used, and then peptides containing arginine are reacted with an unprotected guanidine group.

 The cleavage of the protective Z-groups at intermediate stages is carried out using catalytic hydrogenolysis. As a catalyst, 10% palladium on activated carbon is used.

 In the synthesis of peptides with C-terminal arginine, arginine was introduced into the reaction without protecting the guanidine and carboxyl groups, which significantly simplified the entire synthesis process, since the laborious stages of the introduction and removal of the corresponding protective groups were excluded.

 At the stage of isolation of N-protected peptides with free C-terminal arginine, a purification method was used to avoid repeated reprecipitation, difficulties associated with the close solubility of protected and free arginine-containing peptides. This purification method is silica gel chromatography in the chloroform-methanol (ethanol) system and consists of using specific sorption on silica gel of peptides containing free arginine at the C-terminus.

 All condensations of fragments containing C-terminal free arginine are carried out with the pre-activation of the N-terminal pentapeptide, its conversion to N-hydroxy-5-norbornene-2,3-dicarboxidiimide or pentafluorophenyl ether, which is immediately introduced into the condensation reaction after isolation.

 The final products obtained after removal of the protective groups by acidolytic cleavage with hydrogen bromide in trifluoroacetic acid, or, in the absence of a carbobenzoxy protective group, trifluoroacetic acid, are purified by distribution chromatography or counter current distribution using a planetary centrifuge. For the purification, separation and characterization of peptides and their fragments, HPLC is also used.

 The main problem that arises in the synthesis of analogues of the named natural peptide is the poor solubility of C-terminal fragments.

Therefore, to obtain a undecapeptide with the amino acid sequence Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH, an alternative synthesis scheme is proposed in which the fragmentation is as follows:
(Glp-Pro-Pro-Gly-Gly) + (Ser-Arg) + (Val-Ile-Leu-Phe)
The advantage of this synthesis scheme is that the fragments (Ser-Arg) and (Glp-Pro-Pro-Gly-Gly-Ser-Arg) containing the C-terminal free arginine can be isolated by purification on silica gel as described above.

 The condensation of the fragments (Glp-Pro-Pro-Gly-Gly) and (Ser-Arg) is carried out using the activated ester method (HONB / DCC) with the release of the activated pentapeptide ester, which is immediately used for condensation.

 The final condensation of the fragments (Glp-Pro-Pro-Gly-Gly-Ser-Arg) and (Val-Ile-Leu-Phe) is carried out using diphenylphosphoryl azide.

Upon condensation of the fragments (Glp-Pro-Pro-Gly-Gly-Ser-Arg) and (Val-Ile-Leu-Phe), the activated fragment is the N-terminal fragment having free arginine at the C-terminus. This arginine residue is in the form of an internal salt and does not need additional preparation for activation by adding a base to the reaction mixture. Thus, conducting condensation with diphenylphosphorylazide has another advantage: the absence of an excess of base in the reaction mixture virtually eliminates the possibility of racemization. The yield at this stage was 98%. The identity of this peptide to the peptide obtained by the above method was confirmed by TLC, HPLC and ¹ H-NMR methods.

 The proposed synthesis scheme can serve as the basis for the development of a large-scale synthesis scheme.

The inventive structural analogues of a natural peptide isolated from Hydra attenuata having the following amino acid sequence:
A-Ile-Y-Phe-B,
where A: H-, H-Val-, H-Ser-X-, Glp-Pro-Pro-Gly-Gly-Ser-X-Val;
B: -OH; -Arg-OH;
X: -Lys-, -Arg-;
Y: -Leu-, -Tle-, have biological activity.

 Moreover, the claimed structural analogue, in the general formula of which U represents the remainder —Tie—, has the properties of an inhibitor of growth processes, including pathological processes in mammalian tissues.

 The inventive structural analogues of the named natural peptide, in the general formula of which the substituents A, X and B have the meanings indicated above, and Y represents the remainder -Leu-, have the ability to stimulate the regeneration, growth and differentiation of tissues, as well as stimulate the functions of endocrine and reproductive mammalian systems.

 Studies have been conducted of the inventive analogue of a natural peptide isolated from Hydra attenuata for regeneration processes in the following models: planaria, mealworm larvae, frogs, newts (lens regeneration), rats (liver regeneration, regeneration of skin wounds).

Величина критерия регенерации линейно растет в течение первой недели, а затем темп его роста уменьшается. Анализ динамики пролиферации клеток планарий после перерезки показал ее идентичность с изменением величины критерия регенерации, то есть величина критерия регенерации адекватно отражает процессы регенерации, происходящие в организме.Studies were conducted on the planaria Dugesia tigrina asexual, laboratory animal race. Regeneration was caused by cutting off the front of the planaria with the head ganglion. Immediately after transection, an intensive restructuring of the macromorphological structure of regenerants begins. There is a decrease in the total area and body length with a simultaneous increase in the area and length of the blastema. For the main criterion for regeneration, an indicator of the restoration of the initial proportions of the planar body area was adopted, in particular the ratio of the area of the head part and the entire planar body. This ratio is normalized according to the corresponding indices of intact planarians, and the result is a dimensionless indicator of the regeneration criterion K _pl : B _{o the} total area of the intact planarium, A _{o the} area of the head of the intact planarium, B _{i the} total area of the regenerate on the i-day, A _i the blastema area , m is the number of animals in the group.

The value of the regeneration criterion increases linearly during the first week, and then its growth rate decreases. An analysis of the dynamics of proliferation of planarium cells after transection showed its identity with a change in the value of the regeneration criterion, that is, the value of the regeneration criterion adequately reflects the regeneration processes that occur in the body.

 The specificity of planaria as an experimental model is that the effect of the claimed analogue of a natural peptide isolated from Hydra attenuata on this system is limited to a period of several hours immediately after transection (8-24 hours). When incubating planaria in a solution of the claimed compound, 24 hours after transection, the stimulating effect of this compound is already absent. This is because the peptide acts directly on the cells of the wound surface. At the same time, it should be noted that decapitation of planaria leads to the loss of 1 / 4-1 / 5 of the body part, that is, it is damage at the level of the whole organism. The peptide that entered the body through the wound surface, is included in the endogenous system of regulation of regeneration, which corresponds to the situation of a single injection of the drug into the body of higher animals.

To conduct studies on the effect of the claimed structural analogues of the natural peptide isolated from Hydra attenuata on the regeneration process, it was necessary to establish the optimal concentration range. Immediately after the operation, the planarium was placed in peptide solutions of different concentrations (10 ^-7 - 10 ^-13 M). The inventive compound stimulated regeneration in all experimental groups of animals, however, the peptide showed the greatest effect at concentrations of 10 ^-9 -10 ^-11 M. Therefore, in all subsequent experiments, all compounds were used in this concentration range.

 To test the regeneration process, the intravital morphometry method of planarium regeneration was used. Each experiment was repeated at least three times. To assess the significance of the results, the means were compared using Student's t-test. The ratio of the arithmetic sum of the values of the regeneration criterion on different days of the experiment under the action of a natural compound to the similar amount in the control group under the influence of distilled water was calculated. This ratio was taken as 100%. The corresponding values of this ratio for each of the claimed compounds were compared with this value and expressed as a percentage (% of control).

 The results of the biological activity of structural analogues of the natural peptide isolated from Hydra attenuata are given in Table. 12.

 To emit the influence of the claimed synthesized peptides on regeneration processes in mammals, a model of a full-layer skin wound of the back in rats was used. The experiment was carried out on 89 male Wistar rats, 8-9 animals in one series. Peptides were administered intraperitoneally at a dose of 10 μg / kg in physiological saline 1 time per day for 10 days. The controls were animals that were injected with saline. In the process of wound healing, the dynamics of their contraction (reduction in the area of wounds) and the average time of complete healing (SCH) were evaluated. All results were processed by methods of variation statistics using t-student test. The differences were considered significant at p <0.05. The results of the studies showed that the natural peptide isolated from Hydra attenuata has a significant accelerating effect on the healing of skin wounds. At all measurement dates, the reduction in the area of wounds was significantly ahead of this process in control animals. Under these conditions, the SHIP decreased on average by 6.4 days, which amounted to 27.8% (p <0.01).

The inventive structural analogue of a natural peptide having the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH
practically not inferior to the natural peptide both in accelerating the reduction in the area of wounds and in reducing the CVD (by 22.3% compared with the control).

Thus, a natural peptide isolated from Hydra attenuata and a structural analogue of this natural peptide, which is a compound of the following amino acid sequence:
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH
Being highly active stimulants of regenerative processes, they can be used in surgery for the treatment of sluggish and non-healing wounds, as well as trophic ulcers.

 The effect of the claimed compounds on the anabolic (biosynthetic) processes underlying the regeneration process was investigated. The regenerative rat liver was chosen as an experimental model. The test compounds were administered intraperitoneally at a dose of 20 μg / kg of animal body weight.

 The mammalian liver has the ability to actively proliferate in response to damage. Regenerative liver cells experience significant structural changes already in the early stages after damage. These changes are based on the biosynthesis of RNA, DNA, and protein macromolecules.

On a model of a regenerating liver, a natural peptide isolated from Hydra attenuata, synthesized peptides with the following amino acid sequence were studied:
Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH ([Arg ⁷ ]) and
Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Tle-Phe-OH ([Tle ¹⁰ ])
The biological activity of synthetic analogues of the natural peptide was assessed by their influence on the process of DNA synthesis. The results of the study are given in table. thirteen.

The intensity of incorporation of ³ N-thymidine into the DNA in the liver of partially hepatectomized rats reaches a maximum 22-24 hours after surgery. For biological testing, an interval of 18-22 hours was chosen.

 Thus, based on the data obtained, it can be assumed that the natural peptide isolated from Hydra attenuata and / or its arginine analogue can find clinical application as drugs that stimulate restoration processes in damaged liver tissues. The ability of low doses of these peptides to highly activate the biosynthetic processes underlying the functional and plastic regeneration of the liver, and the absence of such effects on anabolic processes in intact liver tissue makes it possible to consider the named natural peptide and its arginine analogue as promising substances for further medical research.

 Using radioimmunological analysis, the content of a natural peptide isolated from Hydra attenuata was determined in the organs and tissues of mammals. A high peptide content was observed in the pancreas and kidney.

 The hypothalamic localization of the named natural peptide suggests that it can affect growth processes and the reproductive system. In order to verify these assumptions, the effect of the named natural peptide and its analogues on body growth and some hormonal indicators of the functioning of the axis of the hypothalamus-pituitary-gonads were studied. As a result of studying the effect of the named natural peptide and its analogues on the endocrine system, it was found that under the action of the named peptide, the puberty of rats is accelerated. Confirmation of this is an increase in the activity of key enzymes of the metabolism of sex steroid hormones. The introduction of the named natural peptide stimulates an increase in body weight and activates the function of the thyroid gland, which is manifested in a change in a number of morphofunctional indicators (increase in follicle size, number of thyrocytes, etc.).

When studying the effect of analogues of the named peptide on the reproductive and endocrine systems, it was established that [Arg ⁷ ] PP has a pronounced stimulating effect.

 Thus, the named natural peptide acts as a factor accelerating puberty and causing characteristic changes in the functioning of the pituitary gland system.

 From the results of biological testing, it follows that the natural peptide isolated from Hydra attenuata preserves the properties of the regeneration factor in the organisms of higher animals and, in addition, has a specific effect that stimulates the endocrine and reproductive systems.

[Arg ⁷ ] PP is a highly active analogue of the named natural peptide and can be used as a synthetic stimulator of regenerative processes.

[Tle ¹⁰ ] PP is a highly active analogue of the named natural peptide and can be used as an inhibitor of pathological growth processes in mammalian tissues.

 For a better understanding of the claimed invention, the following examples of the method of obtaining the claimed compounds.

 The individuality of the compounds obtained in the following examples was checked by TLC on Kieselgel-60 chromatographic plates (Merck, Germany) in the systems: chloroform methanol acetic acid, 9: 1: 0.5 (A); chloroform methanol 32% acetic acid, 60:45:20 (B); methylene chloride methanol 50% acetic acid, 90: 10: 2 (B); methylene chloride methanol 50% acetic acid, 14: 6: 1 (D); ethyl acetate pyridine acetic acid-water, 45: 20: 6: 11 (D); n-butanol acetic acid water, 3: 1: 1 (E); ethyl acetate - methanol water, 5: 2: 1 (G); chloroform methanol acetic acid, 17: 4.5: 0.6 (3); ethyl acetate hexane, 1: 1 (I); n-butanol acetic acid - pyridine water, 10.5: 6: 1: 7.5 (K); methylene chloride methanol acetic acid, 90: 10: 5 (L); chloroform methanol-acetic acid, 32: 2: 1 (M); isopropyl alcohol ammonia conc. 1: 1 (H); isopropyl alcohol ammonia conc. 2: 1 (0); chloroform methanol ammonia conc. 5: 3: 1 (P), n-butanol pyridine ammonia conc. water, 20: 12: 3: 15 (P).

Solutions of substances in organic solvents after extraction and water washing were dried over anhydrous Na ₂ SO ₄ and evaporated in vacuum on a rotary evaporator at a temperature not exceeding 40 ^o C.

 Peptide lyophilization was performed from frozen aqueous solutions.

 The final products were purified either by countercurrent distribution chromatography in a n: butanol acetic acid water system, 4: 1: 5, the stationary phase was organic, or by reverse phase preparative HPLC on a Gilson chromatograph (France) using a Silasorb RP-18 column "(7.5 μm, 16 x 250 mm) in system A 0.1% aqueous trifluoroacetic acid, B acetonitrile, at a flow rate of 10 ml / min, gradient from O to 60% B in 60 minutes.

Peptide analysis by HPLC was performed on a Gold chromatographic system, Beckman (USA) using an LC-18-DB column (5 μm, 4.6 x 250 mm), Supelco (USA), speed eluent flow 1 ml / min. The analysis was carried out in the following eluent systems:
1. eluent A 0.05% aqueous trifluoroacetic acid; B acetonitrile with the addition of 0.05% trifluoroacetic acid;
2. eluent A 0.02 M phosphate buffer (pH 3.0), B acetonitrile.

Two types of gradient were used:
1. from 5 to 25% B in 20 minutes;
2. from 10 to 50% in 40 minutes.

 The given melting points (not corrected) were determined on a Boetius heating device (GDR), and specific optical rotation on a Perkin-Elmer 241 automatic digital polarimeter (USA), cell length 1 dm.

Hydrolysis of peptides was carried out in 6 N. HCl at 110 ^{° C.} for 24 hours. The hydrolyzate was analyzed either by reverse phase HPLC with a pre-column modification of o-phthalaldehyde, in which case the proline content was not determined, or by an amino acid analyzer.

The individuality of the compounds obtained is confirmed by the proton magnetic resonance method. ¹ H-NMR spectra were obtained on a Bruker WM-50 spectrometer (Germany) with an operating frequency of 500 MHz, at 37 ^° C. Samples for measurements were prepared by dissolving 1 mg of the compound in 0.5 ml of dimethyl sulfoxide-d ₆ . All compounds are in acetate form. Chemical shifts in ¹ H-NMR spectra were measured relative to the internal standard of sodium 2,2-dimethyl-2-silapentane-5-sulfonate. The assignment of signals in the spectra was done using the double resonance method.

Example 1. Z-Leu-Phe-OBu ^t (1).

To a solution of 1.29 g (5 mmol) of H-CI H-Phe-PBut in 10 ml of DMF was added 0.69 ml (5 mmol) of Et ₃ N. The precipitate was filtered off, the filtrate was cooled to -10 ^° C and 1 was added. 93 g (5 mmol) of Z-Leu-ONp in 10 ml of DMF. The reaction mixture was kept for 20 h at 20 ^o C. DMF was evaporated, the residue was dissolved in ethyl acetate, washed with 2% H ₂ SO ₄ , water. The ethyl acetate fraction was evaporated, re-evaporated with isopropanol. The resulting oil was dissolved in methanol and treated with Amberlite IRA-410 in the OH ^{- -} form until disappearance of p-nitrophenol by TLC. The resin was filtered off, washed on the filter with methanol, the filtrate was evaporated and the resulting oil was dried in vacuo to constant weight. 2.30 g (98%) of compound (1) were obtained. R _f 0.79 (A), 0.70 (B), 0.65 (M).

 Example 2. H-Leu-Phe-OH (Ia).

468.6 mg (1.00 mol) of the compound (Ia) obtained in Example 1 was dissolved in 20 ml of trifluoroacetic acid and a stream of dry hydrogen bromide was passed for 45 minutes. The solution was evaporated, the residue was triturated with ether, the precipitate was filtered off, washed on the filter with ether. The resulting material was subjected to HPLC purification. Received 270 mg (97%) of compound (Ia), so pl. 130-132 ^o C, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ + 37.2 ^° (s 1, AcOH), R _f 0.60 (B), 0.50 (D), 0.67 (E). HPLC: 15.94 min (syst. 2, deg. 1).

Example 3. Z-Ile-Leu-Phe-OBu ^t (II).

2.30 g (4.9 mmol) of compound (I), similar to that described in Example 1, was hydrogenated for 1.5 hours in 70 ml of methanol in the presence of 10% Pd on activated carbon (Fluka, Switzerland). The catalyst was filtered off, the filtrate was evaporated, the residue was dissolved in 25 ml of DMF. The solution was cooled to -10 ^° C and 1.73 g (4.6 mmol) of Z-Ile-ONp in 10 ml of DMF was added. The reaction mixture was kept for 20 hours at 20 ^° C. After evaporation, the residue was dissolved in ethyl acetate, washed with 2% H ₂ SO ₄ , water, and evaporated with isopropanol. The resulting oil was dissolved in methanol and treated with amberlite IRA-410 in the OH ^- - form. The resin was filtered off, washed on the filter with methanol. The filtrate was evaporated, the residue was dissolved in ether and precipitated with hexane. Received 2.33 g (87%) of compound (II) with so pl. 149-150 C, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 11.5 ^° (s 1, DMF); R _f 0.78 (A), 0.72 (B), 0.95 (D), 0.57 (M).

 Example 4. H-Ile-Leu-Phe-OH (IIa).

Based on 116 mg (0.20 mmol) of compound (II) obtained in example 3, by the method similar to that indicated in example 2, received 77 mg (98%) of compound (IIa), so pl. 170-175 ^o With, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ + 13.3 ^° (s 1, AcOH), R _f 0.74 (B), 0.55 (D), 0.72 (E). HPLC: 26.39 min. (syst. 1, city 1), 21.94 min (syst. 2, city 1).

Example 5. Z-Val-Ile-Leu-Phe-OBu ^t (III).

1.75 g (3.00 mmol) of compound (II) obtained in Example 3 were hydrogenated in 50 ml of methanol in the presence of 10% Pd / C. The catalyst was filtered off, the filtrate was evaporated, the oil was dissolved in DMF. The solution was cooled to -10 ^° C and 1.06 g (2.85 mmol) of Z-Val-ONp in 10 ml of DMF was added. The reaction mixture was kept for 20 hours at 20 ^° C. After evaporation, the residue was heated in ethyl acetate, the amorphous precipitate formed was filtered off and washed with hexane on the filter. This procedure was repeated twice. Received 1.70 g (88%) of compound (III) with so pl. 220-221 ^o C, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 12.7 ^° (c 1, DMF); R _f 0.76 (A), 0.72 (B). 0.45 (M).

 Example 6. H-Val-Ile-Leu-Phe-OH (IIIa).

Starting from 102 mg (0.15 mmol) of compound (III) obtained in Example 5, 70 mg (95%) of compound (IIIa) were obtained according to a procedure similar to that described in Example 2, so pl. 175-180 ^o With, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ -11.0 ^° (s 1, AcOH), R _f 0.80 (B), 0.58 (D). 0.75 (E). HPLC: 29.33 min (syst. 1, deg. 1), 25.20 min (syst. 2, deg. 1).

Example 7. Z-Lys (Boc) -Val-Ile-Leu-Phe-OBu ^t (IV).

1.89 g (2.8 mmol) of compound (III) obtained in Example 5 were hydrogenated in 50 ml of DMF in the presence of 10% Pd / C. The catalyst was filtered off, the filtrate was cooled to -10 ^° C, and 1.65 g (3.3 mmol) of Z-Lys (Boc) -ONp in 15 ml of DMF was added. It was held for 40 hours at 20 ^° C. After evaporation of the solvent, the residue was heated in ethyl acetate, the amorphous precipitate formed was filtered off, washed with hexane on the filter, and dried. Received 2.40 g (94%) of compound (IV) with so pl. 260 ^o C (with decomposition); [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 15.4 ^° (s 1, DMF); R _f 0.76 (A), 0.72 (B), 0.40 (M).

 Example 8. H-Lys-Val-Ile-Leu-Phe-OH (IVa).

Obtaining was carried out under conditions similar to those specified in example 2 based on 91 mg (0.10 mmol) of compound (IV) obtained in example 7. Received 60 mg (97%) of compound (IVa), so pl. 223-225 ^o C, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 29.0 ^° (s 1, AcOH), R _f 0.68 (B), 0.40 (E), 0.51 (K). HPLC: 27.46 min (syst. 1, deg. 1), 21.54 min (syst. 2, deg. 1).

Example 9. Z-Ser-Lys (Boc) -Val-Ile-Leu-Phe-OBu ^t (V).

2.33 g (2.56 mmol) of compound (IV) obtained in Example 7 were hydrogenated in 70 ml of DMF in the presence of 10% Pd / C. The catalyst was filtered off, the filtrate was cooled to -10 ^° C, and 1.45 g (3.00 mmol) of Z-Ser-OPcp in 8 ml of DMF was added. The reaction mixture was kept for 20 h at 20 ^o C, evaporated, the residue was heated in a mixture of methanol, ethyl acetate, 1: 1. The precipitated amorphous precipitate was filtered off, washed on the filter with hexane, and dried. Received 2.20 g (88%) of compound (V), so pl. 260 ^o With (decomposition), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 18.3 ^° (s 1, DMF); R _f 0.63 (A), 0.94 (B), 0.51 (C), 0.65 (E), 0.85 (K), 0.26 (M). Amino acid analysis: Ser 1.00 (I); Val 0.51 (I); Phe 1.00 (I); Ile 0.49 (I); Leu 1.02 (I); Lys 1.04 (I).

 Example 10. H-Ser-Lys-Val-Ile-Leu-Phe-OH (Va).

100 mg (0.1 mmol) of compound (V) obtained in Example 9 was dissolved in 20 ml of trifluoroacetic acid and a stream of dry HBr was passed for 1 hour. The solution was evaporated, the residue was triturated with ether. The resulting precipitate was filtered off, washed on the filter with ether and dissolved in 50 ml of 10% acetic acid. The solution was treated with DOWEX 50Wx8 ion exchange resin in acetate form until the reaction to Br ^- ions disappeared. The resin was filtered off, washed on the filter with 10% acetic acid, and the filtrate was evaporated. The residue was dissolved in a 1: 1 methanol acetic acid mixture and precipitated with ether. Recrystallized from water. Received 60 mg (85%) of compound (Vb), so pl. > 260 ^o C (with decomposition), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 33 ^° (c 1, AcOH), R _f 0.65 (B), 0.29 (E), 0.50 (K), ¹ H-NMR spectrum of the table. 3. HPLC: 26.96 min (syst. 1, deg. 1), 21.42 min (syst. 2, deg. 1).

 Example 11. Z-Pro-Gly-Gly-OH (VI).

13.31 g (50 mmol) of Z-Gly-Gly-OH was hydrogenated for 2 hours in 200 ml of methanol in the presence of 10% Pd / C, after which 50 ml of 1N was added. The catalyst was filtered off, the filtrate was evaporated, the residue was dissolved in a mixture of DMF with water. To a solution cooled to -10 ^° C, 18.52 g (50 mmol) of Z-Pro-ONp in 40 ml of DMF was added. It was stirred for 20 hours at 20 ^° C. After evaporation, the residue was dissolved in water and extracted three times with ether. The aqueous fraction was acidified with conc. H ₂ SO ₄ to pH 2 and was extracted with n-butanol. The organic fraction was washed three times with water and evaporated. The residue was crystallized from a mixture of ethyl acetate and hexane. Received 16.00 g (88%) of compound (VI) with so pl. 132-133 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 27.5 ^° (s 1, DMF); R _f 0.74 (B), 0.13 (C), 0.64 (D), 0.55 (D).

 Example 12. Z-Pro-Pro-Gly-Gly-OH (VII).

1.14 g (3.0 mmol) of compound (VI) obtained in Example 11 was hydrogenated for 1 h in 50 ml of methanol in the presence of 10% Pd / C, then 3 ml of 1 N NaOH was added. The catalyst was filtered off, the filtrate was evaporated and the residue was dissolved in 10 ml of DMF. To a solution cooled to -10 ^° C, 1.22 g (3.3 mmol) of Z-Pro-ONp in 10 ml of DMF was added. The reaction mixture was kept for 20 hours at 20 ^° C. After evaporation, the residue was dissolved in water and extracted three times with ether. The aqueous fraction was acidified with conc. H ₂ SO ₄ to pH 2 and was extracted with n-butanol. The organic fraction was washed three times with water and evaporated. The residue was crystallized from a mixture of ethyl acetate and hexane, recrystallized from a mixture of ethyl acetate and ether. Received 1.09 g (79%) of compound (VII) with so pl. 46-47 ^o C; a $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 41.1 ^° (s 1, DMF); R _f 0.78 (B), 0.11 (C), 6.60 (D), 0.42 (D).

 Example 13. Z-Glp-Pro-Pro-Gly-Gly-OH (VIII).

921 mg (2 mmol) of compound (VII) obtained in Example 12 was hydrogenated for 1 h with 30 ml of methanol in the presence of 10% Pd / C, then 2 ml of 1 n NaOH was added. The catalyst was filtered off, the filtrate was evaporated and the residue was dissolved in 15 ml of DMF. To a solution cooled to -10 ^° C was added 769 mg (2 mmol) of Z-Gly-ONp in 10 ml of DMF. Maintained for 20 hours at 20 ^° C. After evaporation, the residue was dissolved in water and extracted three times with ether. The aqueous fraction was treated with a DOWEX 50Wx8 ion exchange resin in H ⁺ form. The resin was filtered off, washed on the filter with water, the filtrate was evaporated. The residue was dissolved by heating in a mixture of isopropyl alcohol with ethyl acetate and hexane was added. When the solution was cooled, crystals precipitated. The precipitate was filtered off, washed with hexane, and dried. Received 980 mg (86%) of compound (VIII) with so pl. 118-119 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 69.8 ^° (s 1, DMF); R _f 0.78 (B), 0.04 (C), 0.38 (D), 0.28 (D). Amino acid analysis: Glu 1.00 (1); Gly 2.06 (2). HPLC: 20.97 min (syst. 1, deg. 1) 20.53 min (syst. 2, deg. 1).

 Example 14. Glp-Pro-Pro-Gly-Gly-OH (VIIIa).

57 mg (0.1 mmol) of compound (VIII) obtained in Example 13 was hydrogenated for 1 hour in 10 ml of methanol in the presence of 10% Pd / C. The catalyst was filtered off, the filtrate was evaporated, the residue was dissolved in isopropanol and precipitated with ether. The procedure was repeated twice. The substance is hydroscopic. Dried in a vacuum desiccator over P ₂ O ₅ . Received 35 mg (79%) of compound (VIIIa), so pl. 155-157 ^o C, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ -103.3 ^° (s 1, AcOH). R _f 0.51 (B), 0.31 (H), 0.22 (K). ¹ H-NMR spectrum of Table 2. HPLC: 10.40 min (syst. 1, deg. 2), 10.13 min (syst. 2, deg. 2).

Example 15. Z-Glp-Pro-Pro-Gly-Gly-Ser-Lys (Boc) -Val-Ile-Leu-Phe-OBu ^t (IX).

550 mg (0.55 mmol) of compound (V) obtained in Example 9 was hydrogenated in 50 ml of DMF in the presence of 10% Pd / C. The catalyst was filtered off. 316 mg (0.55 mmol) of the compound (VIII) obtained in Example 13 and 394 mg (2.20 mmol) of HONB (Fluka, Switzerland) were added to the filtrate. The reaction mixture was cooled to -25 ^° C and a solution of 231 mg (1.1 mmol) of DDC in 5 ml of DMF cooled to the same temperature was added with vigorous stirring. The reaction mixture was stirred for 30 minutes at -25 ^° C, then held for 20 hours at 4 ^° C. The precipitate of N, N'-dicyclohexylurea was filtered off, and the filtrate was evaporated. The residue was treated with 2% H ₂ SO ₄ , the precipitated amorphous precipitate was filtered off, washed on the filter with water, ether, and dried. Received 610 mg (98%) of compound (IX), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 93.68 ^° (s 1, AcOH), R _f 0.70 (D), 0.49 (E), 0.57 (H), 0.85 (K).

 Example 16. Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe-OH (X).

690 mg (0.49 mmol) of compound (IX) obtained in Example 15 was treated under conditions similar to those described in Example 10. After treatment with the resin, the filtrate was evaporated and the residue was dissolved by heating in a mixture of isopropyl alcohol and water. The precipitated amorphous precipitate was filtered off, washed on the filter with ether, and dried. Received 460 mg of compound (X), which was subjected to purification on a planetary centrifuge by countercurrent distribution. Received 269 mg (55%) of compound (X); [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 91.8 ^° (c 1, 50% AcOH), R _f 0.49 (B), 0.11 (D), 0.35 (E). Amino acid analysis: Glu 0.98 (1); Ser 0.97 (1); Gly 2.17 (2); Val 0.52 (1); Phe 1.00 (1); Ile 0.52 (1); Leu 1.04 (1); Lys 1.05 (1). ¹ H-NMR spectrum table. 3. HPLC: 27.19 min (syst. 1, deg. 1), 23.76 min (syst. 2, deg. 1).

Example 17. Z ₃ -Arg-Val-Ile-Leu-Phe-OBu ^t (XI).

681 mg (1.0 mmol) of compound (III) obtained in Example 5 was hydrogenated in 30 ml DVF in the presence of 10% Pd / C. The catalyst was filtered off, the filtrate was cooled to -10 ^° C and 733 mg (1.05 mmol) of Z ₃ -Arg-ONp in 5 ml of DMF was added. Maintained for 20 hours at 20 ^o C. The precipitated amorphous precipitate was filtered off, washed on the filter with ether. It was crystallized from ethyl acetate at 4 ^° C. The precipitate was filtered off, washed on the filter with ethyl acetate, hexane, and dried. Received 1.04 g (94%) of compound (XI), so pl. 237-239 ^o C, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 6.1 ^° (s 1, DMF), R _f 0.75 (A), 0.72 (B), 0.63 (G).

Example 18. Z-Ser (Bu ^t ) -Arg-Val-Ile-Leu-Phe-OBu ^t (XII).

884 g (0.80 mmol (of the compound (XI) obtained in Example 17) were hydrogenated in 25 ml of acetic acid in the presence of 10% Pd / C for 20 minutes, the catalyst was filtered off, the filtrate was evaporated, the residue was evaporated with isopropyl alcohol and dissolved in 50 ml of DMF, the solution was cooled to -10 ^{° C.} and 384 mg (0.84 mmol) of Z-Ser (Bu ^t ) -ONB in 5 ml of DMF was added.The reaction mixture was kept for 20 h at 20 ^° C. After evaporation, the residue was crystallized from of isopropyl alcohol.The resulting precipitate was filtered off, washed on a filter with hexane, dried, and 650 mg (83%) of compound (XII) were obtained, mp 227-229 ^o C, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 15.0 ^° (c 1, DMF), R _f 0.46 (A), 0.82 (B), 0.75 (H). H-NMR spectrum table. 4.

Example 19. Z-Glp-Pro-Pro-Gly-Gly-Ser (Bu ^t ) -Arg-Val-Ile-Leu-Phe-OBu ^t (XIII).

294 mg (0.30 mmol) of compound (XII) obtained in Example 18 was hydrogenated in 50 ml of DMF in the presence of 10% Pd / C to give H-Ser (Bu ^t ) -Arg-Val-Ile-Leu- Phe-OBu ^t (XIIa) c R _f 0.47 (B). After removal of the catalyst, 172 mg (0.30 mmol) of the compound (VIII) obtained in Example 13 and 215 mg (1.20 mmol) of HONB were added to the filtrate. The reaction mixture was cooled to -25 ^° C and a solution of 126 mg (0.60 mmol) of DCC in 10 ml of DMF cooled to the same temperature was added with vigorous stirring. Stirred for 30 min at -25 ^o C, then kept 20 h at 4 ^o C and another 20 h at 20 ^o C. According to TLC, two products with R _{f of} 0.65 (D) and 0.74 (D) were detected.

The reaction mixture was evaporated, the residue was dissolved in n-butanol and extracted with 2% acetic acid. The organic fraction was evaporated to 1/3 of the volume and ether was added. The precipitated amorphous precipitate was filtered off and extracted with heating with a mixture of dimethylformamide and isopropyl alcohol. The precipitate formed upon cooling was filtered off and washed with ether on a filter. Received 179 mg (43%) of compound (XIII), R _f 0.65 (D), 0.79 (B), 0.41 (E), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 94.0 ^° (s 1, AcOH).

The mother liquor was evaporated to 1/3 of the volume and precipitated with ether. The precipitate was filtered off, washed on the filter with ether. Received 92 mg of the product with R _f 0.74 (D).

The resulting product was treated with trifluoroacetic acid for 1 h. Trifluoroacetic acid was evaporated, the residue was triturated with ether. The precipitate was filtered off, washed on the filter with ether and dissolved in 50% acetic acid. The solution was treated with DOWEX-50Wx8 ion exchange resin in acetate form. After removing the resin, the aqueous solution was evaporated, the obtained substance was purified by partition chromatography on a Sephadex G-25 column in a 4: 1: 5 butanol-acetic acid water system. Received 17 mg of Ac-Ser-Arg-Val-Ile-Leu-Phe-OH [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 58.6 ^° (s 1, AcOH), R _f 0.70 (B), 0.37 (D), 0.53 (E). Amino acid analysis: Ser 1.04 (1); Arg 1.05 (1); Val 0.51 (1); Phe 1 (1); Ile 0.50 (1); Leu 1.05 (1). HPLC: 28.98 min (syst. 1, deg. 1), 25.63 min (syst. 2, deg. 1).

 Example 20. Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH (XIV).

 179 mg (0.13 mmol) of compound (XIII) obtained in Example 19 was dissolved in trifluoroacetic acid and a stream of dry hydrogen bromide was passed for 40 minutes. The progress of the reaction was monitored by TLC.

After completion of the reaction, trifluoroacetic acid was evaporated, and the residue was treated with ether. The resulting precipitate was filtered off, washed on the filter with ether and dissolved in 50% acetic acid. Next, they were treated with DOWEX-50Wx8 ion exchange resin in acetate form. After removing the resin, the aqueous solution was evaporated to 20 ml and lyophilized. The resulting product was subjected to purification on a planetary centrifuge by countercurrent distribution. Received 77 mg (51%) of compound (XIV). [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 82.7 ^° (s 1, 2% AcOH), R _f 0.60 (B), 0.15 (D), 0.27 (E). Amino acid analysis: Glu 0.93 (1); Ser 0.99 (1); Gly 1.90 (20; Arg 1.02 (1); Val 0.49 (1); Phe 1.0 (1); Ile 0.50 (1); Leu 1.05 (1). HPLC: 27.69 min (syst. 1, deg. 1), 24.28 min (syst. 2, deg. 1). ¹ H-NMR spectrum of Table 5.

Example 21. Z-Ser (Bu ^t ) -Arg-OH (XV).

To a suspension of 0.871 g (5 mmol) of H-Arg-OH in 100 ml of DMF cooled to -25 ^{° C. was} added a solution of 2.511 g (5.5 mmol) of Z-Ser (Bu ^t ) -ONB in 5 ml of DMF. It was stirred for 30 min at -25 ^o C and 24 h at 20 ^o C. DMF was evaporated, the residue was dissolved in 10 ml of chloroform and applied to a chromatographic column (24 x 100 mm) filled with a suspension of silica gel, starting from 25 g of dry silica gel L 100 / 250, Chemapol (Czechoslovakia), in chloroform. Chromatography was performed using a stepwise gradient in a chloroform – ethanol system from 0 to 100% ethanol, increasing the ethanol content in each step by 10%. The eluent volume in each step corresponded to three free column volumes. Detection was performed using a UV spectrophotometric cell "2238 LKB" (Switzerland), wavelength 254 nm. The individuality of the obtained product was determined by TLC. Obtained 2.145 g (95%) of compound (XV), so pl. 124-125 ^o With, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 3.3 ^° (s 1, AcOH), 3.5 (s 1, MeOH), R _f 0.61 (B), 0.48 (E), 0.53 (G).

Example 22. Z-Glp-Pro-Pro-Gly-Gly-Ser (Bu ^t ) -Arg-OH (XVI).

316 mg (0.7 mmol) of the compound (XV) obtained in Example 21 was hydrogenated in 15 ml of methanol in the presence of 10% Pd / C, the catalyst was filtered off, the filtrate was evaporated to 2 ml and precipitated with ether. The precipitate was filtered off, washed on the filter with ether, dried in a vacuum desiccator (the substance was hydroscopic). Received 218 mg (98%) of compound (XVa), R _f 0.22 (B), 0.34 (E).

400 mg (0.7 mmol) of the compound (VIII) obtained in Example 13 and 138 mg (0.77 mmol) of HONB were dissolved in 25 ml of DMF, cooled to -25 ^° and 147 mg (0.7 mmol) of DCC was added. in 2 ml of DMF. The mixture was stirred for 20 minutes at -25 ^° C and 6 hours at 20 ^° C. The precipitated precipitate of N, N'-dicyclohexylurea was filtered off, the solvent was evaporated to 2 ml and precipitated with ether. Received 476 mg (93%) of Z-Glp-Pro-Pro-Gly-Gly-ONB, with R _f 0.70 (B), 0.48 (E), which was immediately dissolved in 5 ml of DMF, cooled to - 25 ^o C and was added to a solution of 205 mg (0.65 mmol) of the previously obtained compound (XVa), in 5 ml of DMF, cooled to the same temperature. The reaction mixture was stirred for 20 minutes at -25 ^° C and 20 hours at 20 ^° C, the solvent was evaporated to 2 ml and precipitated with ethyl acetate. The obtained crystalline precipitate was dissolved in 2 ml of chloroform and applied to a chromatographic column with silica gel. Chromatography was carried out under conditions similar to those described in example 21, the desired product was eluted at 100% methanol. The fraction containing the product was evaporated to 2-3 ml and precipitated with ether. Received 560 mg (99%) of compound (XVI) with so pl. 176-178 ^o C, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 98.3 ^° (s 1, AcOH), R _f 0.60 (B), 0.16 (D), 0.33 (E). HPLC: 25.64 min (syst. 1, deg. 1), 22.45 min (syst. 2, deg. 1), ¹ H-NMR spectrum of the table. 6.

Example 23. Z-Glp-Pro-Pro-Gly-Gly-Ser (Bu ^t ) -Arg-Val-Ile-Leu-Phe-OBu ^t (XIII ').

55 mg (0.10 mmol) of compound (III) obtained in Example 5 was hydrogenated in 10 ml of DMF in the presence of 10% Pd / C. The catalyst was filtered off, the filtrate was evaporated, the residue was dissolved in 2 ml of ethyl acetate and precipitated with hexane. Received 49 mg (90%) of H-Val-Ile-Leu-Phe-OBu ^t (IIIb), R _f 0.22 (A), 0.50 (L), 0.90 (B).

65 mg (0.75 mmol) of the compound (XVI) obtained in Example 22, and 41 mg (0.075 mmol) of the previously obtained compound (IIIb) were dissolved in 5 ml of DMF. The solution was cooled to 0 ^{° C.} and 24 μl of diphenylphosphoryl azide was added with stirring. The reaction mixture was stirred for 6 hours at 0 ^{° C.} and 24 hours at 20 ^{° C.} , DMF was evaporated to 2 ml and precipitated with ethyl acetate. The precipitate was filtered off, washed on the filter with ethyl acetate, ether. The resulting precipitate was redissolved under heating in 5 ml of DMF and 2 ml of trifluoroethanol and precipitated with ether. Received 103 mg (98%) of compound (XIII), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 93.90 ^° (s 1, AcOH), R _f 0.77 (B), 0.65 (D), 0.44 (E). Amino acid analysis: Glu 0.98 (1), Ser 0.76 (1), Arg 1.06 (1), Gly 1.99 (2), Val 0.77 (1), Ile 0.79 (1) , Leu 1.02 (1), Phe 1.00 (1).

Example 24. H-Tle-Phe-OBu ^t (XVII).

To a solution of 776 mg (3.0 mmol) of H-Phe-OBu ^t HCl in 5 ml of DMF was added 0.41 ml (3.0 mmol) of Et ₃ N. The precipitate was filtered off, the filtrate was cooled to -10 ^° C and a solution was added. 1.280 g (3.3 mmol) of Z-Tle-ONp in 5 ml of DMF. The reaction mixture was held for 48 hours at 20 ^{° C.} , 41 mg (0.3 mmol) of HOBt was added and left for another 20 hours. The solution was evaporated and the residue was dissolved by heating in ethyl acetate. Upon cooling, unreacted H-Phe-OBut precipitated, which was filtered off and the mother liquor was washed with 2% H ₂ SO ₄ , water. The ethyl acetate fraction was evaporated, the residue was dissolved in methanol and treated with DOWEX-1 ion exchange resin in the OH form until p-nitrophenol disappeared according to TLC. The resin was filtered off, washed on the filter with methanol, the filtrate was evaporated to 10 ml and subjected to hydrogenation in the presence of 10% Pd / C. The catalyst was filtered off and the filtrate was evaporated. The obtained H-Tle-Phe-OBu ^t (XVII) dipeptide was purified on a Lobar chromatographic column (310 25) LiChroprep Si 60 (40 63 μm) Merck (Germany). Chromatography was performed in isocratic mode; system (A) was used as eluent. The fraction containing the desired product was evaporated and lyophilized from an aqueous solution. Received 465 mg (47%) of compound (XVII); t. pl. 104-105 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ 23.5 ^° (s 1, DMF), 31 ^o (s 1, AcOH), R _f 0.35 (A), 0.73 (D), 0.68 (E), 0.80 (G).

Example 25. Z-Ile-Tle-Phe-OBu ^t (XVIII).

To a solution of 435 mg (1.3 mmol) of compound (XVII) obtained in Example 24 in 5 ml of DMF cooled to 10 ^{° C. was} added a solution of 502 mg (1.3 mmol) of Z-Ile-ONp in 2 ml of DNF . The reaction mixture was kept for 20 hours at 20 ^{° C.} , then the solution was evaporated to 1/3 of the volume and precipitated with ether. The resulting precipitate was dissolved in methanol and treated with DOWEX-1 ion-exchange resin in the OH form until HONp disappeared according to TLC. The resin was filtered off, washed on the filter with methanol, the methanol was evaporated. The resulting oil was crystallized from a methanol-water mixture. Received 608 mg (81%) of compound (XVIII); so pl. 85-86 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 18 ^° (s 1, AcOH); R _f 0.76 (I), 0.90 (W), 0.55 (M).

Example 26. Z-Val-Ile-Tle-Phe-OBu ^t (XIX).

291 mg (0.5 mmol) of the compound (XVIII) obtained in Example 25 was hydrogenated in 5 ml of methanol in the presence of 10% Pd / C, the catalyst was filtered off, the filtrate was evaporated, the residue was dissolved in 10 ml of DMF and cooled to -10 ^{° C.} To the resulting solution was added 205 mg (0.55 mmol) of Z-Val-ONp in 2 ml of DMF and held for 20 hours at 20 ^° C. The solution was evaporated, the oil was dissolved in ether and precipitated with hexane. The resulting precipitate was recrystallized from methanol-water. Received 245 mg (72%) of compound (XIX); so pl. 205-206 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 34 ^° (s 1, AcOH); R _f 0.63 (I), 0.85 (G), 0.43 (M).

Example 27. Z-Lys (Boc) -Val-Ile-Tle-Phe-OBu ^t (XX).

204 mg (0.3 mmol) of compound (XIX) obtained in Example 26 was hydrogenated in 10 ml of DMF in the presence of 10% Pd / C. The catalyst was filtered off, the filtrate was cooled to -10 ^{° C.} and 165.5 mg (0.33 mmol) of Z-Lys (Boc) -ONp was added to it, left for 24 hours at 20 ^° C. The solution was evaporated, the residue was dissolved in ether and besieged with hexane. The procedure was repeated twice. Received 125 mg (47%) of compound (XX); so pl. 229-230 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 25 ^° (c 1, AcOH); R _f 0.25 (I), 0.86 (G), 0.78 (A), 0.41 M).

Example 28. Z-Ser-Lys (Boc) -Val-Ile-Tle-Phe-OBu ^t (XXI).

98 mg (0.11 mmol) of compound (XX) obtained in Example 27 was hydrogenated in methanol in the presence of 10% Pd / C. The catalyst was filtered off, the filtrate was evaporated, the residue was dissolved in DMF and cooled to -10 ^° C. 63 mg (0.13 mmol) of Z-Ser-OPcp was added to the solution. It was held for 20 h at 20 ^° C, DMF was evaporated, the residue was dissolved in a mixture of methanol and ether and precipitated with hexane. The precipitate was separated by filtration, chloroform ether-hexane was dissolved in a mixture and allowed to crystallize for 20 hours at 4 ^° C. The resulting crystalline precipitate was filtered off and washed on a filter with hexane. Received 93 mg (85%) of compound (XXI); mp 231-232 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 33 ^° (s 1, AcOH); R _f 0.70 (A), 0.90 (G). 0.30 (M). Amino acid analysis: Ser 0.98 (1), Lys 1.02 (1), Val 0.56 (1), Ile 0.60 (1), Phe 1.00 (1). ¹ H-NMR spectrum table. 7.

Example 29. Z-GLp-Pro-Pro-Gly-Gly-Ser-Lys (Boc) -Val-Ile-Tle-Phe-OBu ^t (XXII).

85 mg (0.085 mmol) of compound (XXI) obtained in Example 28 was hydrogenated in DMF in the presence of 10% Pd / C to give H-Ser-Lys (Boc) -Val-Ile-Leu-Phe-OBu ^t ( XXIa) c R _f 0.80 (B). After removal of the catalyst, 49 mg (0.085 mmol) of the compound (VIII) obtained in Example 13 was added to the solution. The mixture was cooled to -25 ^° C and a solution of 244 mg (0.26 mmol) of “complex F” was added under vigorous stirring, cooled to the same temperature. The reaction mixture was kept for 30 minutes at -25 ^° C, then 20 hours at 4 ^° C and 24 hours at 20 ^° C. The N, N'-dicyclohexylurea precipitate was filtered off, the mother liquor was evaporated, the residue was dissolved in 3 ml of DMF and precipitated with ether. Received 95 mg (79%) of compound (XXII); so pl. > 260 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 72 ^° (c 1, AcOH); R _f 0.72 (D), 0.51 (E), 0.59 (H).

 Example 30. Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Tle-Phe-OH (XXIII).

91 mg (0.06 mmol) of compound (XXII) obtained in Example 29 was dissolved in trifluoroacetic acid and dry HBr flow was passed for 45 minutes, then the solution was evaporated, the oil was triturated with ether, the precipitate was filtered off and washed on the filter with ether. The resulting material was purified by preparative HPLC. Received 60 mg (89%) of compound (XXIII); so pl. 260 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 81 ^° (s 1, AcOH); R _f 0.52 (B), 9.23 (E), 0.45 (K). Amino acid analysis: Ser 0.80 (1), Glu 0.95 (1), Pro 2.05 (2), Gly 2.05 (2), Val 0.90 (1), Ile 0.91 (1) , Phe 1.00 (1), Lys 0.91 (1). HPLC: 26.15 min (syst. 1, deg. 1), 22.82 min (syst. 2, deg. 1).

 Example 31. Z-Phe-Arg-OH (XXIV).

To a suspension of 1.74 g (10 mmol) of H-Arg-OH in 20 ml of DMF was added 5.04 g (12 mmol) of Z-Phe-ONp and stirred for 20 hours at 20 ^° C. The DMF was evaporated, the residue was dissolved in 25 ml of chloroform and subjected to chromatographic purification under conditions similar to those described in example 21. Received 4.3 g (94%) of compound (XXIV); t. pl. 131-132 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 15.2 ^° (s 1, DMF); R _f 0.70 (B), 0.50 (E), 0.63 (K).

 Example 32. Z-Leu-Phe-Arg-OH (XXV).

4.25 g (9.3 mmol) of compound (XXIV) obtained in Example 31 was hydrogenated in 50 ml of methanol in the presence of 10% Pd / C for 2 hours, R _f 0.38 (B) of compound H-Phe- Arg-OH (XXIVa). The catalyst was filtered off, washed on the filter with methanol, the filtrate was evaporated and the residue was dissolved in 25 ml of DMF. 3.86 g (10 mmol) of Z-Leu-ONp was added to the solution cooled to -10 ^{° C.} It was held for 30 min at -10 ^° C and left for 20 h at 20 ^° C. Further processing and chromatography was carried out as described in procedure (XXIV). Received 4.75 g (90%) of compound (XXV); so pl. 146-147 ^o C, [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 13.3 ^° (s 1, AcOH); R _f 0.82 (B), 0.43 (D), 0.56 (E).

 Example 33. Z-Ile-Leu-Phe-Arg-OH (XXVI).

4.55 g (8.00 mmol) of the compound (XXV) obtained in Example 32 was hydrogenated in 100 ml of methanol in the presence of 10% Pd / C, R _f 0.50 (B) H-Leu-Ile-Phe-Arg -OH (XXVa). The catalyst was filtered off and the filtrate was evaporated. To the residue, dissolved in 25 ml of DMF and cooled to -10 ^{° C.} , 3.4 g (0.80 mmol) of Z-Ile-ONp in 5 ml of DMF was added. Maintained for 24 hours at 20 ^o C. The solvent was evaporated and the residue was subjected to chromatographic purification, similar to that described in example 21, while the desired product was eluted only when the column was washed with ethanol acetic acid, 1: 2. The fraction containing the desired product was evaporated and the resulting oily residue was crystallized from methanol at 4 ^° C. The obtained crystalline precipitate was filtered off, washed on the filter with ether, dried over NaOH in a desiccator. Received 4.35 g (96%) of compound (XXVI); so pl. 189-190 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 21.0 ^° (s 1, AcOH), R _f 0.85 (B), 0.45 (D), 0.60 (E).

 Example 34. Z-Val-Ile-Leu-Phe-Arg-OH (XXVII).

2.05 g (3.00 mmol) of compound (XXVI) obtained in Example 33 was hydrogenated in a mixture of DMF H ₂ O, 1: 1, in the presence of 10% Pd / C. The catalyst was filtered off, the filtrate was evaporated, the residue was dissolved in 30 ml of DMF under heating. A solution of 1.23 g (3.30 mmol Z-Val-ONp in 5 ml DMF was added to the cooled solution. The reaction mixture was stirred 48 hours at 20 ^° C. Ether was added to the precipitated amorphous precipitate and the resulting precipitate was filtered. Trifluoroethanol was recrystallized twice from the mixture. methanol, 1: 1. Received 2.00 g (85%) of compound (XXVII); mp 247-248 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 28.5 ^° (s 1, AcOH); R _f 0.86 (B), 0.48 (D), 0.62 (E).

 Example 35. Z-Lys (Boc) -Val-Ile-Leu-Phe-Arg-OH (XXVIII).

1.56 g (2.00 mmol) of compound (XXVII) obtained in Example 34 was hydrogenated in acetic acid in the presence of 10% Pd / C. The catalyst was filtered off, washed on the filter with acetic acid, and the filtrate was evaporated. The residue was re-evaporated several times with water. Crystallized from methanol at 4 ^o C. Dried over NaOH in a desiccator. Obtained 1.23 g (95%) of H-Val-Ile-Leu-Phe-Arg-OH (XXVIIa), R _f 0.48 (B). HPLC: 24.01 min (syst. 1, deg. 1), 18.07 min (syst. 2, deg. 1).

0.65 g (1.00 mmol) of the title compound (XXVIIa) was dissolved in a 3: 1 DMF DMSO mixture, cooled to -10 ^° C and 0.58 g (1.15 mmol) of Z-Lys (Boc) - was added. ONp. It was stirred for 48 hours at 20 ^° C. The solvent was evaporated to 3 ml and ether was added. The resulting precipitate was filtered off, washed with ether on a filter. Recrystallized twice from trifluoroethanol. Received 0.57 g (57%) of compound (XXVIII); so pl. 260 ^o C (with decomposition); [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 19.0 ^° (with 1, 75% AcOH); R _f 0.88 (B), 0.55 (D), 0.66 (E).

 Example 36. Z-Ser-Lys (Boc) -Val-Ile-Leu-Phe-Arg-OH (XXIX).

0.71 g (0.70 mmol) of compound (XXVIII) obtained in Example 35 was hydrogenated in acetic acid in the presence of 10% Pd / C. The catalyst was filtered off, washed on the filter with acetic acid, the filtrate was evaporated, and the residue was several times evaporated with water. Crystallized from a mixture of isopropyl alcohol with hexane. The resulting precipitate was dried over NaOH in a vacuum desiccator. Received 0.57 g (93%) of H-Lys- (Boc) -Val- -Ile-Leu-Phe-Arg-OH (XXVIIIa), R _f 0.64 (B). HPLC: 35.37 min (syst. 1, grad. 1).

0.57 g (0.65 mmol) of the title compound (XXVIIIa) was dissolved in a 3: 1 DMF DMSO mixture, cooled to -10 ^° C and 0.37 g (0.72 mmol) of Z-Ser-OPcp was added. The reaction mixture was stirred 48 hours at 20 ^° C. The solvent was evaporated, the residue was dissolved in 5 ml of DMF with heating and precipitated with ether. The procedure was repeated twice. The precipitate was filtered off, washed on the filter with ether. Received 0.55 g (77%) of compound (XXIX); [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 54.5 ^° (with 1, 50% AcOH); R _f 0.80 (B), 0.59 (E), 0.64 (W), 0.70 (K), 0.40 (3). Amino acid analysis: Ser 0.85 (1), Arg 0.98 (1), Val 0.88 (1), Ile 0.79 (1), Leu 1.01 (1), Lyz 0.99 (1) , Phe 1.00 (1).

 Example 37. Z-Glp-Gly-Gly-Pro-Pro-Ser-Lys (Boc) -Val-Ile-Leu-Phe-Arg-OH (XXX).

300 mg (0.27 mmol) of the compound (XXIX) obtained in Example 36 was hydrogenated in acetic acid in the presence of 10% Pd / C. The catalyst was filtered off, washed on the filter with acetic acid, and the filtrate was evaporated. The residue was re-evaporated several times with water and n-butanol. Crystallized from water. Received 250 mg (95%) of H-Ser-Lys (Boc) -Val-Ile-Leu-Phe-Arg-OH (XXIXa), R _f 0.48 (B), HPLC: 33.81 min (syst. 1, city 1). 27.46 min (syst. 2, deg. ¹ ) ¹ H-NMR spectrum of the table. eight.

54 mg (0.094 mmol) of the compound (VIII) obtained in Example 13 was dissolved in 2 ml of DMF, cooled to -25 ^{° C.} and 270 mg (0.282 mmol) of “complex F” in 1.5 ml of DMF were added with stirring. It was stirred for 30 minutes at -25 ^° C, 24 hours at 2 ^° C. The precipitated precipitate of N, N'-dicyclohexylurea was filtered off, the filtrate was evaporated to 1/3 of the volume and precipitated with ether. Received 73 mg (96%) of Z-Glp-Pro-Pro-Gly-Gly-Opcp, R _f 0.85 (B), which immediately after precipitation was dissolved in 2 ml of DMF, cooled to -25 ^o C and added with stirring to a solution of 85 mg (0.90 mmol) of the previously obtained compound (XXIXa) in a mixture of 3 ml of DMF and 4 ml of hexamethylphosphoramide (Fluka). Stirred for 30 minutes at -25 ^° C, 72 hours at 20 ^° C. DMF was evaporated, the residue was precipitated with ether. Received 128 mg (94%) of compound (XXX), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 94.0 ^° (s 1, AcOH), R _f 0.73 (B), 0.35 (E), 0.75 (O), 0.43 (P).

 Example 38. Glp-Gly-Gly-Pro-Pro-Ser-Lys-Val-Ile-Leu-Phe-Arg-OH (XXXI).

115 mg (0.076 mmol) of the compound (XXX) obtained in Example 37 was dissolved in 25 ml of trifluoroacetic acid and a stream of dry HBr was passed for 1 hour. The solution was evaporated, the residue was triturated with ether and filtered. Received 113 mg (98%) of compound (XXXI), which was subjected to purification by HPLC. The yield of purified product was 95 ml (98%), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 81.25 ^° (s 1, AcOH), R _f 0.40 (B), 0.16 (E), 0.80 (H), 0.22 (O), 0.30 (P). Amino acid analysis: Glu 1.01 (1), Ser 0.86 (1), Arg 1.07 (1), Gly 2.05 (2), Pro 2.03 (2), Val 0.79 (1), Ile 0.82 (1), Leu 1.02 (1), Lyz 1.02 (1), Phe 1.00 (1). HPLC: 23.69 min (syst. 1, deg. 1), 19.05 min (syst. 2, deg. 1), ¹ H-NMR spectrum of the table. 9.

Example 39. Z ₃ -Arg-Val-Ile-Leu-Phe-Arg-OH (XXXII).

To a suspension of 253 mg (0.4 mmol) of the compound (XXVIIa) obtained in Example 35. in 10 ml of DMF was added a solution of 335 mg (0.48 mmol) of Z ₃ -Arg-ONp in 2 ml of DMF and the reaction mixture was stirred for 48 hours at 20 ^o C. To the precipitated amorphous precipitate was added ether and filtered. Reprecipitated from DMF several times with ether. Received 268 mg (56%) of compound (XXXII); so pl. 242-244 ^o C; [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 24.3 ^° (s 1, AcOH); R _f 0.90 (B), 0.50 (D), 0.55 (E).

Example 40. Z-Ser (Bu ^t ) -Arg-Val-Ile-Leu-Phe-Arg-OH (XXXIII).

241 mg (0.20 mmol) of the compound (XXXII) obtained in Example 39 was hydrogenated in acetic acid in the presence of 10% Pd / C for 3 hours. The catalyst was filtered off, the filtrate was evaporated and the residue was precipitated from methanol with ether. The resulting precipitate was dried in a vacuum desiccator over NaOH. Received 157 mg (98%) of H-Arg-Val-Ile-Leu-Phe-Arg-OH (XXXIIa), R _f 0.15 (B). HPLC: 23.26 min (syst. 1, deg. 1), 16.00 min (syst. 2, deg. 1).

145 mg (0.18 mmol) of compound (XXXIIa) was dissolved in a 2: 1 DMF DMSO mixture, cooled to -10 ^{° C.} and 99 mg (0.22 mmol) of Z-Ser (Bu ^t ) -ONB was added. Stirred for 48 hours at 20 ^° C. The solution was evaporated to 90% of the volume and precipitated with ether. The precipitate was filtered off, washed on the filter with ether, dried and dissolved by heating in 3 ml of DMF. The amorphous precipitate that formed upon cooling was filtered off, washed on the filter with chilled DMF, ether, and dried. The procedure was repeated twice. Received 130 mg (67%) of compound (XXXIII); so pl. 253-256 ^o With (decomposition), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 43 ^° (s 1, AcOH), R _f 0.66 (B), 0.31 (D), 0.44 (E). Amino acid analysis: Ser 0.79 (1), Arg 2.12 (2), Val 0.78 (1), Ile 0.80 (1), Leu 1.02 (1), Phe 1.00 (1) .

 Example 41. lp-Pro-Pro-Gly-Gly-Ser (Bu) -Arg-Val-Ile-Leu-Phe-Arg-OH (XXXIV).

108 mg (0.1 mmol) of compound (XXXIII) obtained in Example 40 was hydrogenated in acetic acid in the presence of 10% Pd / C, and isolated under conditions similar to those described in Example 40. 95 mg (100%) of H- was obtained. Ser (Bu ^t ) -Arg-Val-Ile-Leu-Phe-Arg-OH (XXXIIIa), R _f 0.67 (H), 0.48 (K), 0.30 (D).

44 mg of Glp-Pro-Pro-Gly-Gly-OH was dissolved in 15 ml of DMF, cooled to -25 ^° C and a solution of "complex F" in 5 ml of DMF was added with stirring. The precipitated precipitate of N, N'-dicyclohexylurea was filtered off, the solvent was evaporated to 1/3 of the volume and precipitated with ether. The precipitate was filtered off and dissolved in 2 ml of DMF. The resulting solution was cooled to -10 ^{° C.} and 95 mg of compound (XXXIIIa) in 20 ml of DMF was added to a solution. 13.5 mg (0.1 mmol) of HOBt was added to the solution. The mixture was stirred for 48 hours at 20 ^° C. The solvent was evaporated, the residue was dissolved in methanol and precipitated with ethyl acetate. Received 90 mg (70%) of compound (XXXIV), R _f 0.45 (H).

 Example 42. Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-Arg-OH (XXXV).

90 mg (0.07 mmol) of compound (XXXIV) obtained in Example 41 was dissolved in trifluoroacetic acid and kept for 1 h at 20 ^° C. Trifluoroacetic acid was evaporated, the residue was triturated with ether, filtered and washed with ether on a filter. Purified by HPLC. Received 78 mg (85%) of compound (XXXV), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 60 ^° (c 1, AcOH); R _f 0.43 (B), 0.32 (H), 0.75 (H), 0.18 (P). Amino acid analysis: Glu 1.01 (1), Ser 0.75 (1), Arg 2.03 (2), Gly 1.95 (2), Pro 1.89 (2), Val 0.79 (1) , Ile 0.83 (1), Leu 1.02 (1), Phe 1.00 (1). HPLC: 24.36 min (syst. 1, deg. 1), 19.82 min (syst. 2, deg. 1). ¹ H-NMR spectrum table. 10.

 Example 43. H-Ser-Arg-Val-Ile-Leu-Phe-Arg-OH (XXXIIIb).

Was obtained by HPLC purification obtained in example 42, the compound in an amount of 10% of the initial amount of compound (XXXIIIa) similar to that specified in example 41. Received 9 mg (XXXIIIb), [a] $\genfrac{}{}{0ex}{}{\text{22}}{\text{D}}$ - 26 ^° (s 1, AcOH), R _f 0.38 (B), 0.75 (N). HPLC: 23.15 min (syst. 1, deg. 1). 16.59 min (syst. 2b grad. ¹ ), ¹ H-NMR spectrum of the table. eleven.

Sources of information taken into account:
1. Birr C. Zachmann B. Bodenmuller H. Shcaller HC "Synthesis of a new neuropeptide, the head activator from hydra", Febs. Lett. 1981, V. 131, N 1, p. 317-321.

Claims (7)

1. Derivatives of the peptides of General formula I
A Ile Y Phe B,
where A H-, H- Val -, H Ser X Val -, Glp Pro Pro Gly Gly - Ser X Val -;
B-OH, -Arg OH, X LyS -, -Arg -;
Y Leu -, -Tle,
where is Tle L-tert-leucine. 2. The derivative of the peptide according to claim 1, which is a undecapeptide of the following amino acid sequence
Glp Pro Pro (Gly), Ser Arg Val Ile Leu Phe OH. 3. The derivative of the peptide according to claim 1, which is a undecapeptide of the following amino acid sequence
Glp Pro Pro Gly Gly Ser Lys Val Ile Tle Phe OH. 4. The derivative of the peptide according to claim 1, which is a peptide of the following amino acid sequence
Glp Pro Pro Gly Gly Ser Lys Val Ile Tle Phe Arg - OH. 5. The derivative of the peptide according to claim 1, which is a peptide of the following amino acid sequence
Glp Pro Pro (Gly) ₂ Ser Arg Val Ile Tle Phe Arg OH. 6. A method of obtaining derivatives of peptides of General formula I
A Ile Y Phe B,
where A H-, H Val -, H Ser X Val -, Glp Pro Pro Gly Gly - Ser X Val -;
B OH, Arg OH, X Lys -, -Arg -;
Y Leu -, Tle -,
characterized in that the peptide chain is sequentially extended from the C-terminus of the amino acid sequence using an activated ester of an alpha-amino protected amino acid and the resulting compound of formula A Ile Y Phe B,
where A: H-, H Val -, H Ser X Val -,
the values of B, X and Y are indicated above,
if necessary, in the case where A H Ser X Val - is subjected to fragment condensation in the presence of dicyclohexylcarbodiimide and an anti-racemization additive with the corresponding N-terminal peptide to obtain compound I
where A H- Glp Pro Pro Gly Gly Ser X Val. 7. A method of obtaining derivatives of peptides of General formula I
A Jle Leu Phe OH, where A HGlp-Pro-Pro-Gly-Gly- Ser-Arg-Val-,
characterized in that the peptide chain is sequentially extended, starting from the C-end of the fragment, to obtain the N-terminal pentapeptide: Glp Pro Pro Gly Gly, Ser Arg dipeptide and Val-Ile-Leu Phe C-terminal tetrapeptide, and the resulting peptide fragments by sequentially attaching the Ser Arg dipeptide to the N-terminal pentapeptide with dicyclohexylcarbodiimide in the presence of a racemization inhibitor and attaching the Glp Pro Pro Gly Gly Ser Arg C-terminal tetrapeptide to the obtained N-terminal heptapeptide Val Ile Leu Phe with diphenylphosphorylazide.

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