Derivatives of peptides and method of production thereof

 

(57) Abstract:

Usage: in medicine and biology, in particular in the method of obtaining derivatives of peptides. The inventive product - derived peptides f-ly 1: A-Ile-Y-Phe-B, where a Is N, HVal, Hser-X-Val, Glp-Pro-Pro-Gly-Glg-Ser-X-Val: B - OH, Arg-OH: X-Lys. Arg: Y - Leu, Tle. Reagent 1: C-terminal amino acid. Reaction conditions: 1) a coherent extension of the peptide chain by using activated esters protected alpha-amino group of amino acids with the formation of compounds f-ly 1, where A - H, HVal, HSer-X-Val, which are subjected to a fragmented condensation in the presence of dicyclohexylcarbodiimide and excluding racemization supplementation with N-terminal connection: Glp-Pro-Pro - Gly-Gly-Ser-X-Val; 2) obtaining derivatives of peptides f-crystals: A-Ile-Y-Phe-OH, where A - Glp-Pro-Pro-Gly-Gly-Ser-X-Val, are consistent extension of the peptide chain, starting from the C-end fragment, to obtain the N-terminal Pentapeptide: Glp-Pro-Pro-Gly-Gly, the dipeptide Ser-Arg and C-terminal tetrapeptide: Val-Ile-Leu-Phe, followed by condensation of the obtained fragments consistent adherence to these N-terminal Pentapeptide of the dipeptide Ser-Arg c by using dicyclohexylcarbodiimide in the presence of excluding racemization supplements and joining the resulting hexopal.

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

The invention can find application in the creation of new highly effective medication.

It is now established that in the brain tissue of a primitive chordate animals presented more than a dozen peptides identical to the cortex of the human brain. The active study of these peptides has practical value, since the identification of specific functions phylogenetically ancient peptides of the higher animals can serve as a basis for creation of new highly effective medication.

One of the known phylogenetically ancient peptides is a natural peptide isolated from Hydra attenuata. This substance is undecapeptide with a molecular mass of 1124 and amino acid sequence

Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe-OH.

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

of man; I believe that these peptide can act as a growth factor in the normal development of higher animals and humans, and can also regulate the body's functions in various pathological conditions.

For research to clarify the biological functions called undecapeptide isolated from Hydra attenuata, requires a significant amount of this substance, which can only be obtained by chemical synthesis, Natural analogues of this peptide was not detected, and all of the synthesized analogues do not possess the biological activity of the natural peptide.

By chemical synthesis were obtained following peptides [1] (1) lp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe-OH (2) H-Tyr-Cln-Pro-Pro-Cly-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 performed by a solid phase method, as a protective group for aminophenol used a, a-dimethyl-3,5-dimethoxybenzonitrile group, to block e-amino group of lysine used benzyloxycarbonyloxy protection, to protect the hydroxyl group Arina and tyrosine used tertbutylphenol group, 4,4-dimethoxybenzonitrile protective group used in the case of glutamine.

After removal from the 1 output of 18% (2) yield 47%, and (3) to exit 33%

The compounds obtained were loirevalley one peak under conditions of highly effective liquid chromatography (HPLC) with this release of the peptide (1) coincide with the time of the native peptide. Joint chromatography of these two peptides, synthetic and natural, also gave one peak.

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

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

The basis of the claimed invention is the task to create new substances

structural analogues of the natural peptide isolated from Hydra attenuata with Biologicheskaya and mammalian tissues, and inhibitors of pathological growth processes in mammalian tissues, as well as ways to obtain such structural analogues.

This task is solved in that the biologically active structural analogue of the 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, making manifest the properties of the control processes of regeneration and tissue growth, and regulator of the endocrine system.

An embodiment of the claimed invention is that biologically active structural analogue of the natural peptide isolated from Hydra attenuata, is undecapeptide following amino acid sequence:

Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH,

which shows properties of synthetic stimulator of regeneration of tissues and organs.

Another variant of the invention consists in that the biologically active structural analogue of the natural peptide isolated from Hydra attenuata is undecapeptide following amino acid sequence:

Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Tle-Phe-OH,

the claimed invention is what biologically active structural analogue of the natural peptide isolated from Hydra attenuata is a Hexapeptide following amino acid sequence:

H-Ser-X-Val-Ile-Leu-Phe-OH, X: -Lys-, -Arg-,

which shows the properties of the stimulator of regeneration processes.

An embodiment of the claimed invention is that biologically active structural analogue of the natural peptide isolated from Hydra attenuata is tetrapeptide following amino acid sequence:

H-Val-Ile-Y-Phe-OH, Y-Leu-,

which shows the properties of the stimulator of regeneration processes.

An embodiment of the claimed invention is that biologically active structural analogue of the natural peptide isolated from Hydra attenuata, is a peptide of the following amino acid sequence:

Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-LEU-Lle-Phe-Arg-OH

or the following amino acid sequence:

Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-LEU-Lle-Phe-Arg-OH,

which have the ability to stimulate the regeneration of tissues and organs.

In accordance with the claimed invention, suitable biologically active structural analogues of the natural peptide isolated from the second sequence with the activated ester of the amino acids, protected alpha-amino groups, in which, according to the invention, the gradual increase in the peptide chain is carried out until obtaining peptide:

A-Ile-Y-Phe-B

where A: H-, H-Val, H-Ser-X;

B: -OH, -Arg-OH;

X: -Lys-, -Arg-;

Y: -Leu, -Tle,

followed by fragment condensation with dicyclohexylcarbodiimide in the presence of additives, excluding racemization, to obtain a peptide of the following sequence:

A-Ile-Y-Phe-B

where A: Glp-Pro-Pro-Gly-Gly-Ser-X-Val-,

In, X and Y have the above values.

In accordance with the claimed invention, it is expedient upon receipt of the peptide 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-LEU-Lle-Phe-Arg-OH or

Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-LEU-Lle-Phe-Arg-OH,

to use the way in which the gradual increase in the peptide chain carried out to obtain N-terminal Pentapeptide 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 -,

X-Lys-, -Arg-; Y-Leu-, -Tle-; B: -OH, -Arg-OH.

In accordance with the claimed invention, it is expedient if the receiving item is about, in which sequential build-up of the peptide chain carried out to obtain N-terminal Pentapeptide following amino acid sequence of Glp-Pro-Pro-Gly-Gly, the dipeptide Ser-Arg and C-terminal tetrapeptide Val-Ile-Leu-Phe, and the condensation of the resulting peptide fragments is carried out by sequential accession to the above-mentioned N-terminal Pentapeptide of the dipeptide Ser-Arg using dicyclohexylcarbodiimide in the presence of additives, excluding racemization, and by joining the N-terminal heptapeptide Glp-Pro-Pro-Gly-Gly-Ser-Arg C-terminal tetrapeptide Val-Ile-Leu-Phe with diphenylphosphinite.

Further objectives and advantages of the invention will become apparent from the subsequent detailed description of the biologically-active structural analogues of the natural peptide isolated from Hydra attenuata, the retrieval method, specific examples of the claimed compounds and experimental tests of the claimed compounds.

In accordance with the claimed invention, the features of biologically active structural analogue of the natural peptide isolated from Hydra attenuata, representing 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-G the s data of amino acid analysis1H-NMR spectroscopy and mass spectroscopy.

The obtained compounds were characterized by the value of the specific rotation []2D2, melting point, data thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC).

The claimed peptide compounds get classical methods of peptide chemistry in solution using, if necessary, fragment condensation.

So, for example, upon receipt of 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 peptide with the following amino acid sequence:

Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-LEU-Lle-Phe-Arg-OH

or peptide with the following amino acid sequence

Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-LEU-Lle-Phe-Arg-OH,

perform sequential build-up of the peptide chain from the C-end amino acid sequence of the fragment to obtain the N-terminal Pentapeptide following amino acid sequence:

Glp-Pro-Pro-Gly-Gly

and C-terminal peptide with the following amino acid sequence:
Westlaw with the activated ester of the amino acids, protected alpha-amino group, for example, n-nitrophenylamino ether, pentachlorphenol ester, N-hydroxy-5-norbornene-2,3-dicarboximide ether carbonintensity.

The next stage of obtaining these undecapeptide is condensation obtained by successive extensions of the peptide chain fragments before the formation of the target product, the condensation is carried out using dicyclohexylcarbodiimide in the presence of additives, excluding racemization, for example, in the presence of N-hydroxy-5-Narbonne-2,3 - dicarboximide.

The sequential extension of the peptide chain in accordance with this methodology, the excess activated ester and emitted from the reaction of n-NITROPHENOL or NORCR or N removed with a solvent in which the main reaction product is insoluble, i.e. presidenial. In some cases, when the solubility of the desired product, p-NITROPHENOL was close, for example C-terminal di - and tri-peptides, p-NITROPHENOL is removed by treatment with America IRA-410 in the form while using TLC.

The combination of protective groups largely determines the conditions for further synthesis. For example, the use of N-go, when a stepwise increase of the peptide chain from the C-end method of activated esters and the use of protective urethane type condensation are not accompanied by racemizations.

The combination of protective groups depends, in turn, the specific amino acid sequence and is determined by the possibility of selective release of temporary and permanent protective groups. Because of the structural analogues of the natural peptide isolated from Hydra attenuata, no serosoderzhanie amino acids, as a temporary protective group selected benzyloxycarbonyl group, tsepliaeva by hydrogenolysis, as permanent protective groups using group tretbutylphenol type, resistant to hydrogenolysis and otsepleniya acidolysis. This combination of protective groups used in the synthesis of fragments on the above methodology: as a temporary protection of the amino group using benzyloxycarbonyl protective group (Z), for permanent blocking of e-amino group of lysine using tert-butyloxycarbonyl protective group (BOC), phenylalnine introduced into the reaction in the form of tert-butyl methyl ether, to protect a hydroxyl function of serine using tert-Buti is W ith minimum protection of functional groups. So carboxyl group of the C-terminal glycine for the synthesis of a fragment of Glp-Pro-Pro-Gly-Gly protect the salt formation, the hydroxyl group of serine 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

left unprotected, and in other cases, using tert-boutelou protection.

Upon receipt of the peptides with Arg in the 7-position, 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 joining arginine use p-nitrophenyloctyl broadcast tricarb benzoxa arginine, and further, the peptides containing arginine, enter into reaction with the unprotected guanidino group.

The removal of protective Z-groups at intermediate stages is carried out using catalytic hydrogenolysis. As catalyst, use 10% palladium on charcoal.

In the synthesis of peptides with C-terminal arginine, arginine was introduced into the reaction without the protection of guanidino and carboxyl groups that znachitelno simplified the whole process of synthesis, were excluded because labor-intensive stages of introducing and removing the protective groups.

At the stage of selection of the N-protected peptides with a free C-terminal arginine was used method is Semenych and free argininemia peptides. This treatment method is a chromatography on silica gel in the system chloroform-methanol (ethanol) and consists in the use of specific sorption on silica gel peptides containing-the end of free arginine.

All the condensation of fragments containing the C-terminal free arginine, carried out with predictively N-terminal Pentapeptide, converting it into N-hydroxy-5-norbornene-2,3-dicarboximide or pentafluorophenyl ester, which immediately after the separation is introduced into the reaction of condensation.

The final products obtained after removal of the protective groups algoritmicheskom splitting of hydrogen bromide in triperoxonane acid, or, in the absence of carbobenzoxy protective group, triperoxonane acid, was subjected to purification methods distribution chromatography or countercurrent distribution using the planetary centrifuge. For purification, separation and characterization of peptides and their fragments is also used HPLC.

The main problem encountered in the synthesis of analogues of these natural peptide is the poor solubility of C-terminal fragments.

Therefore, to obtain undecapeptide with amino acid after the fragments is as follows:

(Glp-Pro-Pro-Gly-Gly) + (Ser-Arg) + (Val-Ile-Leu-Phe)

The advantage of this scheme of synthesis is that the fragments (Ser-Arg) and (Glp-Pro-Pro-Gly-Gly-Ser-Arg), containing C-terminal free arginine, can be identified with the help of purification on silica gel described above.

Condensation of fragments (Glp-Pro-Pro-Gly-Gly) and (Ser-Arg) is conducted by the method of activated esters (HONB/DCC) with the release of the activated ester Pentapeptide, which is immediately used for the condensation.

The final condensation of fragments (Glp-Pro-Pro-Gly-Gly-Ser-Arg) and (Val-Ile-Leu-Phe) is performed using diphenylphosphinite.

By condensation of fragments (Glp-Pro-Pro-Gly-Gly-Ser-Arg) and (Val-Ile-Leu-Phe) - activated fragment is the N-terminal fragment having a free arginine at the C-end. This arginine residue is in the form of internal salts, and does not need additional preparation for activation of the addition of base in the reaction mixture. Thus, carrying out the condensation with diphenylphosphinite has another advantage: the absence of an excess of base in the reaction mixture virtually eliminates the possibility of racemization. The output of this stage was 98% Identity of this peptide the peptide obtained in the above way, confirmed by TLC, HPLC and1N-Amami synthesis.

The claimed structural analogues of the 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, possess biological activity.

While the claimed structural analogue, in the General formula which represents the balance-Tie-, has the properties of an inhibitor of growth processes, including pathological processes in mammalian tissues.

The claimed structural analogues of these natural peptide, in the General formula of which the substituents A, X and b have the values specified above, and represents the balance-Leu-have the ability to stimulate the regeneration, growth and differentiation of tissues, and to stimulate the functions of the endocrine and reproductive systems of mammals.

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

Studies were conducted on the planaria Dugesia tigrina gleam. Immediately after transection begins intensive rebuilding micromorphologically patterns regenerants. Is the reduction of the total area and length of the body with a simultaneous increase in the size and length of blastema. The main criterion regeneration was adopted indicator restore the original proportions of the square body of planaria, in particular the ratio of the area of the head part and the whole body of planaria. This ratio is normalized by the corresponding indicators of intact planaria, and the result is a dimensionless measure of the criterion regeneration TOPL: INothe total area of the intact planaria ANDothe area of the head part of the intact planaria, INithe total area of the regenerate at the i-th day, ANDisquare blastema, m the number of animals in the group.

The value of the criterion regeneration is increased linearly during the first week, and then the growth rate decreases. Analysis of the dynamics of cell proliferation of planaria after transection showed its identity with the magnitude of the criterion of regeneration, that is, the value of the criterion regeneration adequately reflects the regeneration processes occurring in the body.

The specificity of planaria as an experimental model is that the effect of the proposed analogue of dstone after transection (8-24 h). When the incubation of planaria in the solution of the claimed compounds after 24 h after transection stimulating effect of this compound have been missing. This is due to the fact that the peptide acts directly on cells of the wound surface. However, it should be noted that the decapitation of planaria leads to the loss of 1/4-1/5 part of the body, then there is damage at the level of the whole organism. Peptide, trapped in the body through the wound surface, is involved in the endogenous regulation system regeneration, which corresponds to the situation of a single injection of the drug into the body of higher animals.

For conducting research on the effects of the claimed structural analogues of the natural peptide isolated from Hydra attenuata, in the regeneration process, it was necessary to establish the optimal concentration range. Immediately after the operation of planaria were placed in solutions of different peptide concentrations (10-7- 10-13M). The claimed connection stimulated regeneration in all experimental groups of animals, however, the greatest effect of the peptide was shown at concentrations of 10-9-10-11M. Therefore, in all subsequent experiments, all compounds used in this concentration range.

For testrow the UNT was repeated at least three times. To assess the significance of the results was performed comparing the average values using t-student criterion. Calculate the ratio of the arithmetic sum of the values of the criterion regeneration on different days of experience under the action of natural compounds similar to the amount in the control group, which was under the action of distilled water. This ratio was taken as 100%, the Corresponding value of this relationship for each of the claimed compounds were compared with this value and expressed in percent (% from control).

The results of the biological activity of structural analogues of the natural peptide, you divided from Hydra attenuata, are given in table. 12.

For radiation effects claimed synthesized peptides on the processes of regeneration in mammals was used to model panolayou skin wound back in rats. The experiment was carried out in 89 rats male Wistar rats, 8-9 animals in one series. The peptides were administered intraperitoneally at a dose of 10 µg/kg in saline 1 time a day for 10 days. Control served as the animals, which were injected with saline. In the process of wound healing was assessed by the dynamics of their contraction (shrinking RAS) and average duration of complete healing (GCC). Su and considered differences at p < 0,05. The results of these studies showed that the natural peptide isolated from Hydra attenuata has a significant accelerating effect on the healing of skin wounds. On all terms of measuring the reduction in the area of RAS was significantly ahead of the process control animals. Under these conditions, the GCC has fallen to an average of 6.4 days, which was 27.8% (p < 0,01).

The claimed structural analogue of the natural peptide having the following amino acid sequence:

Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH

almost as good as the natural peptide as in accelerating the reduction in the area of wounds and reduce GCC (22.3% compared with control).

Thus, a natural peptide isolated from Hydra attenuata and structural analogue of this natural peptide that represents the connection sleboda amino acid sequence:

Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH

being highly active promoters of regenerative processes, can be used in surgery for the treatment of indolent and long-term healing of wounds and trophic ulcers.

We studied the effect of the claimed compounds in anabolic (biosynthetic) processes underlying the process regeneratively intraperitoneally, at a dose of 20 μg/kg animal body weight.

The mammalian liver has the ability to actively proliferative growth in response to damage. Cell regenerating liver are experiencing significant structural changes at early time points after injury. At the heart of these changes are the processes of biosynthesis of macromolecules RNA, DNA, and proteins.

On the model of regenerating liver were investigated natural peptide isolated from ydra attenuata synthesized peptide with the following amino acid sequence:

Glp-Pro-Pro-Gly-Gly-Ser-Arg-Val-Ile-Leu-Phe-OH ([Arg7])

Glp-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Tle-Phe-OH ([Tle10])

Evaluation of biological activity of synthetic analogues of natural peptide were carried out to provide their influence on the process of DNA synthesis. The results of the study are given in table. 13.

Intensity include3H-thymidine into DNA in the liver partially hepatectomized rats reaches its maximum after 22-24 h after surgery. For biological testing was selected interval 18-22 h

Thus, on the basis of the obtained data it can be assumed that the natural peptide isolated from Hydra attenuata and/or arginine analogue can find clinical p. the ability of low doses of these peptides in a high degree to activate biosynthetic processes, the underlying functional and plastic regeneration of the liver, and the absence of such effects on anabolic processes in the intact liver tissue provides an opportunity to consider these natural peptide and arginine analogue as promising for further medical research substances.

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

Hypothalamic localization of these natural peptide suggests that it may influence the processes of growth and reproductive system. To verify these assumptions investigated the effects of these natural peptide and its analogues on the growth of the body and some hormonal indicators of functioning axis the hypothalamic-pituitary-sex glands. The study of the influence of these natural peptide and its analogues on the endocrine system, it was found that under the action of these peptide accelerates puberty in rats. This is confirmed by the increased activity of key ferments body and activates the function of the thyroid gland, resulting in a change of the number of functional performance (increase the size of the potentially number of thyrocytes and others).

When studying the effect of analogues of the above mentioned peptide on sexual and endocrine systems established that [Arg7] PP has a pronounced stimulating effect.

Thus, these natural peptide acts as a factor that accelerates puberty and causes characteristic changes in the functioning of the system pituitary sex glands.

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

[Arg7] PP is a highly active analogue of these natural peptide and can be used as a synthetic stimulant regenerative processes.

[Tle10] PP is highly similar to these natural peptide and can be used as an inhibitor of pathological growth processes in mammalian tissues.

The individuality of the compounds obtained in the following examples, was checked by TLC on chromatographic plates Kieselgel-60 ("Megs", Germany) systems: chloroform, methanol, acetic acid, 9:1:0.5 in(A); chloroform methanol 32% acetic acid, 60:45:20(B); methylene chloride methanol, 50% acetic acid, 90:10:2(In); methylene chloride methanol, 50% acetic acid, 14:6:1(G); the ethyl acetate pyridine acetic acid-water, 45:20:6: 11(L); n-butanol acetic acid-water 3:1:1(E); ethyl acetate - methanol-water, 5: 2:1(W); chloroform methanol acetic acid, 17:4,5:0,6(3); the ethyl acetate hexane, 1: 1(And); n-butanol acetic acid - pyridine-water, 10,5:6:1:7,5 (); 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 and pyridine ammonia conc. water, 20:12:3:15(R).

Solutions of substances in organic solvents after extraction and the aqueous washings were dried over anhydrous Na2SO4and was evaporated in vacuo on a rotary evaporator at a temperature not exceeding 40oC.

The lyophilization of the peptides was performed from frozen aqueous solutions.

The final products were purified Leonarda the organic phase, either method obremeniaet preparative HPLC on a chromatograph "Gilson" (France), using the column "Silasorb RP-18" (7.5 μm, 16 x 250 mm) in the system And 0.1% aqueous triperoxonane acid In acetonitrile, at a flow rate of 10 ml/min, gradient from About to 60% In 60 min

Analysis of peptides by HPLC was performed on a chromatographic system "Gold," the company "Beckman", (USA) using column LC-18-DB (5 µm, 4.6 x 250 mm), the company "Supelco", (USA), the flow rate of eluent, 1 ml/min Analysis was performed on the following systems eluents:

1. eluent And 0.05% of water triperoxonane acid In acetonitrile with the addition of 0.05% triperoxonane acid;

2. eluent And 0.02 M phosphate buffer (pH 3.0), In acetonitrile.

Were used two types of gradient:

1. from 5 to 25% In 20 min;

2. from 10 to 50% at 40 minutes

Above the melting temperature (not adjusted) was determined on the heating device "Boetius" (GDR), and the specific optical rotation automatic digital polarimeter "Perkin-Elmer 241 (USA), the length of the cuvette 1 inch

Hydrolysis of peptides was carried out in 6 BC NS at 110oC for 24 h analysis of the hydrolysate was carried out either by obremeniaet HPLC using a modification of the o-phthalic aldehyde, in this spoluchenych compounds was confirmed by the method of proton magnetic resonance. Spectra1H-NMR of the obtained spectrometer "WM-50" "Bruker (Germany) with an operating frequency of 500 MHz at 37oC. Samples for measurements were prepared by dissolving 1 mg of the compound in 0.5 ml of DMSO-d6. All connections in the acetate form. Chemical shifts in the spectra of 1H-NMR measured relative to the internal standard 2,2-dimethyl-2-silapentane-5-sulfonate sodium. The assignment of signals in the spectra is made using the double resonance.

Example 1. Z-Leu-Phe-OBut(1).

To a solution of 1.29 g (5 mmol) H-CI H-Phe-PBut in 10 ml of DMF was added to 0.69 ml (5 mmol) Et3N. the precipitation was filtered, the filtrate was cooled to -10oWith and added 1,93 g (5 mmol) of Z-Leu-ONp in 10 ml of DMF. The reaction mixture was stirred for 20 h at 20oC. DMF was evaporated, the residue was dissolved in ethyl acetate, washed with 2% N2SO4, water. An ethyl acetate fraction was evaporated, persuasively with isopropanol. The resulting oil was dissolved in methanol and treated with amberlite IRA-410 in OH-form to the disappearance of p-NITROPHENOL according to TLC. The resin was filtered, washed on the filter with methanol, the filtrate was evaporated and the oil obtained was dried in vacuum until constant weight. Received 2,30 g (98%) of compound (1). Rf0,79 (A) 0,70 (B) Of 0.65 (M)in 20 ml triperoxonane acid and passed a current of dry hydrogen bromide for 45 minutes The solution was evaporated, the residue triturated with ether, the precipitate was filtered, washed on the filter with ether. The resulting material was subjected to HPLC purification. Obtained 270 mg (97% ) of compound (Ia), so pl. 130-132oC [a]2D2+ 37,2(1, Asón), Rf0,60 (B), AND 0.50 (D) OF 0.67 (E). HPLC: 15,94 min (Syst. 2, deg. 1).

Example 3. Z-Ile-Leu-Phe-OBut(II).

2.30 g (4.9 mmol) of the compound (I), similar to that specified in example 1, was first made 1.5 h in 70 ml of methanol in the presence of 10% Pd on charcoal ("Fluka, Switzerland). The catalyst was filtered, the filtrate was evaporated, the residue was dissolved in 25 ml of DMF. The solution was cooled to -10oWith and was added 1.73 g (4.6 mmol) of Z-LEU-ONp in 10 ml of DMF. The reaction mixture was stirred for 20 h at 20oC. After evaporation the residue was dissolved in ethyl acetate, washed with 2% N2SO4water was evaporated with isopropanol. The resulting oil was dissolved in methanol and treated with amberlite IRA-410 in the HE-- form. The resin was filtered, washed on the filter with methanol. The filtrate was evaporated, the residue was dissolved in ether and precipitated with hexane. Received of 2.33 g (87%) of compound (II) with so pl. 149-150, [a]2D2- 11,5(C 1, DMF); Rf0,78 (A) TO 0.72 (B) OF 0.95 (D) 0,57 (M).

Example 4. H-Ile-Leu-shown in example 2, received 77 mg (98%) of compound (IIa), so pl. 170-175oC [a]2D2+ 13,3(1, Asón), Rf0,74 (B) OF 0.55 (D) 0,72 (E). HPLC: 26,39 minutes (Syst. 1, deg. 1), 21,94 min (Syst. 2, deg. 1).

Example 5. Z-Val-Ile-Leu-Phe-OBut(III).

of 1.75 g (3.00 mmol) of compound (II) obtained in example 3, was first made in 50 ml of methanol in the presence of 10% Pd/C catalyst was Filtered, the filtrate was evaporated and the oil was dissolved in DMF. The solution was cooled to -10oWith and added 1.06 g (2,85 mmol) Z-Val-ONp in 10 ml of DMF. The reaction mixture was stirred for 20 h at 20oC. After evaporation the residue was heated in ethyl acetate, the resulting amorphous precipitate was filtered and washed on the filter with hexane. This procedure was repeated twice. Received 1.70 g (88%) of compound (III) with so pl. 220-221oC [a]2D2- 12,7(c 1, DMF); Rf0,76 (A) 0,72 (IN). 0,45 (M).

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

On the basis of 102 mg (0.15 mmol) of the compound (III) obtained in example 5 by the method similar to the one mentioned in example 2 were obtained 70 mg (95%) of compound (IIIa), so pl. 175-180oC [a]2D2-11,0(1, Asón), Rf0,80 (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-Ovii 10% Pd/C. The catalyst was filtered, the filtrate was cooled to -10oWith and added 1.65 g (3.3 mmol) of Z-Lys(Boc)-ONp in 15 ml of DMF. Kept for 40 h at 20oC. After evaporation of the solvent the residue was heated in ethyl acetate, the resulting amorphous precipitate was filtered, washed on the filter with hexane, and dried. Received 2,40 g (94%) of the compound (IV) with so pl. 260oC (decomposition); [a]2D2- 15,4(C 1, DMF); Rf0,76 (A) 0,72 (IN), AND 0.40 (M).

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

Obtaining carried out in similar conditions specified in example 2, on the basis of 91 mg (0.10 mmol) of the compound (IV) obtained in example 7. Received 60 mg (97%) of compound (IVa), so pl. 223-225oC [a]2D2- 29,0(1, Asón), Rf0,68 (B), AND 0.40 (E) 0,51 (K). HPLC: 27,46 min (Syst. 1, deg. 1), 21,54 min (Syst. 2, degree 1).

Example 9. Z-Ser-Lys(Boc)-Val-Ile-Leu-Phe-OBut(V).

of 2.33 g (2.56 mmol) of the compound (IV) obtained in example 7, was first made in 70 ml of DMF in the presence of 10% Pd/C Catalyst was filtered, the filtrate was cooled to -10oWith and added 1.45 g (3.00 mmol) of Z-Ser-OPcp in 8 ml DMF. The reaction mixture was stirred for 20 h at 20oWith, was evaporated, the residue was heated in a mixture of methanol, ethyl acetate, 1:1. Precipitated amorphous precipitate hoteltravel the P>2D2- 18,3(C 1, DMF); Rf0,63 (A) 0,94 (B) 0,51 (B) OF 0.65 (E) 0,85 (K) 0,26 (M). Amino acid analysis: Ser 1,00 (I); Val 0,51 (I); Phe 1.00 and (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 the compound (V) obtained in example 9, was dissolved in 20 ml triperoxonane acid and passed a current of dry Nug for 1 h, the Solution was evaporated, the residue triturated with ether. The precipitate was filtered off, washed on the filter with ether, and dissolved in 50 ml of 10% acetic acid. The solution was treated with ion exchange resin DOWEX 50Wx8 in acetate form to the disappearance of the reaction ions VG-. The resin was filtered, washed on the filter with 10% acetic acid, the filtrate was evaporated. The residue was dissolved in a mixture of methanol, acetic acid, 1:1 and precipitated with ether. Recrystallized from water. Received 60 mg (85%) of compound (V), so pl. > 260oC (with decomposition), [a]2D2- 33(c 1, Asón), Rf0,65 (B) 0,29 (E), AND 0.50 (K)1H NMR spectrum 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 first made 2 hours in 200 ml of methanol in the presence of 10% Pd/C, after which was added 50 ml of 1 N. the Catalyst was filtered and Phil who mmol) of Z-Pro-ONp in 40 ml of DMF. Was stirred 20 h at 20oC. After evaporation the residue was dissolved in water and was extracted three times with ether. The aqueous fraction was acidified using conc. H2SO4to pH 2 and extracted with n-butanol. The organic fraction is washed three times with water, was evaporated. The residue was led from a mixture of ethyl acetate with hexane. Received 16,00 g (88%) of compound (VI) with so pl. 132-133oC; [a]2D2- 27,5(C 1, DMF); Rf0,74 (B) 0,13 (B) OF 0.64 (G) OF 0.55 (D).

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

1,14 g (3.0 mmol) of the compound (VI) obtained in example 11, was first made 1 h in 50 ml of methanol in the presence of 10% Pd/C was then added 3 ml of 1 n NaOH. The catalyst was filtered, the filtrate was evaporated and the residue was dissolved in 10 ml of DMF. To a cooled to -10oTo the solution was added 1.22 g (3.3 mmol) of Z-Pro-ONp in 10 ml of DMF. The reaction mixture was stirred for 20 h at 20oC. After evaporation the residue was dissolved in water and was extracted three times with ether. The aqueous fraction was acidified using conc. H2SO4to pH 2 and extracted with n-butanol. The organic fraction is washed three times with water, was evaporated. The remainder crystallizable from a mixture of ethyl acetate with hexane, and recrystallized from a mixture of ethyl acetate and ether. Received 1,09 g (79%) of compound (VII) with so pl. 46-R 13. Z-Glp-Pro-Pro-Gly-Gly-OH (VIII).

921 mg (2 mmol) of the compound (VII) obtained in example 12, was first made 1 hour and 30 ml of methanol in the presence of 10% Pd/C was then added 2 ml of 1 n NaOH. The catalyst was filtered, the filtrate was evaporated and the residue was dissolved in 15 ml of DMF. To a cooled to -10oTo the solution was added 769 mg (2 mmol) of Z-Gly-ONp in 10 ml of DMF. Kept for 20 h at 20oC. After evaporation the residue was dissolved in water and was extracted three times with ether. The aqueous fraction was treated with ion exchange resin DOWEX 50Wx8 in the N+form. The resin was filtered, washed on the filter with water, the filtrate was evaporated. The residue was dissolved under heating in a mixture of isopropyl alcohol with ethyl acetate and hexane was added. Upon cooling of the solution precipitated crystals. The precipitate was filtered, washed with hexane, and dried. Received 980 mg (86%) of the compound (VIII) with so pl. 118-119oC; [a]2D2- 69,8(C 1, DMF); Rf0,78 (B) 0,04 (B) 0,38 (G), AND 0.28 (D). Amino acid analysis: Glu 1.00 each (1); Gly to 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 the compound (VIII) obtained in example 13, was first made 1 h in 10 ml of methanol in the presence of 10% Pd/C Catalyst was filtered, the filtrate was evaporated, the residue rhum-desiccator over P2ABOUT5. Received 35 mg (79%) of compound (VIIIa), so pl. 155-157oC [a]2D2-103,3(1, Asón), Rf0,70 (D) 0,49 (E) 0,57 (C), 0,85 ().

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

690 mg (0.49 mmol) of the compound (IX) obtained in example 15, were treated in the same terms as specified in example 10. After treatment with the resin, the filtrate was evaporated and the residue was dissolved under heating in a mixture of isopropyl alcohol and water. Precipitated amorphous precipitate was filtered, washed on the filter with ether, dried. Received 460 mg of the compound (X), which was subjected to purification on a planetary centrifuge method countercurrent distribution. Received 269 mg (55%) of the compound (X); [a]2D2- 91,8(c 1, 50% of the Asón), Rf0,49 (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 each (1); Ile 0,52 (1); Leu 1,04 (1); Lys 1.05 (1).1H NMR spectrum table. 3. HPLC: 27,19 min (Syst. 1, deg. 1), 23,76 min (Syst. 2, deg. 1).

Example 17. Z3-Arg-Val-Ile-Leu-Phe-OBut(XI).

681 mg (1.0 mmol) of the compound (III) obtained in example 5 was first made in 30 ml of DVF in the presence of 10% Pd/C Catalyst was filtered, the filtrate was cooled to -10oWith and added 733 mg (1.05 mmol) of Z3-Arg-ONp in 5 ml of DMF. Vyderjala of ethyl acetate at 4oC. the Precipitate was filtered, washed on the filter with ethyl acetate, hexane, and was dried. Obtained 1.04 g (94%) of compound (XI), so pl. 237-239oC [a]2D2- 6,1(C 1, DMF), Rf0,75 (A) 0,72 (IN) 0,63 (W).

Example 18. Z-Ser(But)-Arg-Val-Ile-Leu-Phe-OBut(XII).

884 g (0.80 mmol( compound (XI) obtained in example 17, was first made in 25 ml of acetic acid in the presence of 10% Pd/C for 20 minutes, the Catalyst was filtered, the filtrate was evaporated, the residue persuasively with isopropyl alcohol and dissolved in 50 ml of DMF. The solution was cooled to -10oWith and added 384 mg (0.84 mmol) of Z-Ser(But)-ONB in 5 ml of DMF. The reaction mixture was stirred for 20 h at 20oC. After evaporation the residue crystallizable from isopropyl alcohol. The precipitate was filtered off, washed on the filter with hexane, and dried. Received 650 mg (83%) of compound (XII), so pl. 227-229oC [a]2D2- 15,0(c 1, DMF), Rf0,46 (A) 0,82 (B) 0,75 (C). H NMR spectrum table. 4.

Example 19. Z-Glp-Pro-Pro-Gly-Gly-Ser(But)-Arg-Val-Ile-Leu-Phe-OBut(XIII).

294 mg (0.30 mmol) of the compound (XII) obtained in example 18, was first made in 50 ml of DMF in the presence of 10% Pd/C, to obtain the compounds H-Ser(But)-Arg-Val-Ile-Leu-Phe-OBut(XIIa) c Rf0,13, and 215 mg (1.20 mmol) of HONB. The reaction mixture was cooled to -25oAnd with vigorous stirring solution was added 126 mg (0,60 mmol) of DCC in 10 ml of DMF cooled to the same temperature. Was stirred for 30 min at -25oWith, then kept for 20 h at 4oAnd another 20 h at 20oC. According to TLC revealed two products with Rf0,65 (D) and 0.74 (D).

The reaction mixture was evaporated, the residue was dissolved in n-butanol and was extracted with 2% acetic acid. The organic fraction was evaporated to 1/3 volume and added ether. Precipitated amorphous precipitate was filtered and was extracted by heating a mixture of dimethylformamide with isopropyl alcohol. Precipitated upon cooling, the precipitate was filtered, washed on the filter with ether. Received 179 mg (43%) of compound (XIII), Rf0,65 (D) 0,79 (B) 0,41 (E) [a]2D2- 94,0(1, Asón).

The mother liquor was evaporated to 1/3 volume and precipitated with ether. The precipitation was filtered, washed on the filter with ether. Received 92 mg of the product with Rf0,74 (D).

The obtained product was treated triperoxonane acid for 1 h Triperoxonane acid was evaporated, the residue triturated with ether. The precipitation was filtered, washed on the filter, the air is After removal of the resin aqueous solution was evaporated, the resulting substance was purified by means of distributing chromatography on a column of Sephadex G-25 in the system butanol acetic acid-water, 4:1:5. Received 17 mg of Ac-Ser-Arg-Val-Ile-Leu-Phe-OH [a]2D2to 58.6(1, Asón), Rf0,70 (B), AND 0.37 (D) OF 0.53 (E). Amino acid analysis: Ser 1,04 (1); Arg of 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 the compound (XIII) obtained in example 19 was dissolved in triperoxonane acid and passed a current of dry hydrogen bromide for 40 minutes Over the course of the reaction was monitored using TLC.

After the reaction triperoxonane acid was evaporated, the residue was treated with ether. The precipitate was filtered off, washed on the filter with ether, and dissolved in 50% acetic acid. Next was treated with ion exchange resin DOWEX-50Wx8 in acetate form. After removal of the resin aqueous solution was evaporated to 20 ml and was liofilizovane. The obtained product was subjected to purification on a planetary centrifuge method countercurrent distribution. Received 77 mg (51% ) of compound (XIV). [a]2D2- 82,7(1, 2% Asón), Rf0,60 (B) 0,15 (D), 0,27 (E). An amino acid is happy. 1).1H NMR spectrum table.5.

Example 21. Z-Ser(But)-Arg-OH (XV).

To a suspension 0,871 g (5 mmol) of H-Arg-OH in 100 ml of DMF, cooled to -25oTo that solution was added 2,511 g (5.5 mmol) of Z-Ser(But)-ONB in 5 ml of DMF. Was stirred for 30 min at -25oC and 24 h at 20oC. DMF was evaporated, the residue was dissolved in 10 ml of chloroform and applied to the chromatographic column (24 x 100 mm), filled with a slurry of silica gel on the basis of 25 g of dry silica gel L 100/250, "Chemapol" (Czechoslovakia), in chloroform. Chromatography was produced using a stepwise gradient in the system chloroform - ethanol from 0 to 100% ethanol, increasing the ethanol content in each stage by 10% the Volume of eluent in each stage corresponded to the three free column volumes. Detection was performed using UV spectrophotometric cell 2238 LKB (Switzerland), wavelength 254 nm. The individuality of the obtained product was determined by TLC. Received 2,145 g (95%) of compound (XV), so pl. 124-125oC [a]2D2- 3,3(1, Asón), 3,5 (1, Meon), Rf0,61 (B) 0,48 (E) OF 0.53 (W).

Example 22. Z-Glp-Pro-Pro-Gly-Gly-Ser(But)-Arg-OH (XVI).

316 mg (0.7 mmol) of the compound (XV) obtained in example 21, was first made in 15 ml of methanol in the presence of 10% PD/C catalyst ochiltree in vacuumassisted (substance gigroskopichna). Received 218 mg (98%) of compound (XVa), Rf0,22 (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 -25oand added 147 mg (0.7 mmol) of DCC in 2 ml of DMF. Was stirred 20 min at -25oC and 6 h at 20oC. the precipitation of N, N'-dicyclohexylmethane was filtered, the solvent was evaporated to 2 ml and precipitated with ether. Received 476 mg (93%) of Z-Glp-Pro-Pro-Gly-Gly-ONB, with Rf0,70 (B) 0,48 (E), which was immediately dissolved in 5 ml of DMF, cooled to -25oWith and added to a solution of 205 mg (of 0.65 mmol) previously obtained compound (XVa), in 5 ml of DMF, cooled to the same temperature. The reaction mixture was stirred 20 min at -25oC and 20 h at 20oC, the solvent was evaporated to 2 ml and precipitated with ethyl acetate. The resulting crystalline precipitate was dissolved in 2 ml of chloroform and applied to a chromatographic column with silica gel. Chromatography was performed in conditions similar to those specified in example 21, the desired product was elyuirovaniya with 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. 176-178oC [a]2D2- 98,3(1, Asón), Rf0,60 (B), 0,16 (D), 0,33 (E). HPLC: 25,64 min (Syst. 1, deg. 1)UP>t(XIII').

55 mg (0.10 mmol) of the compound (III) obtained in example 5 was first made in 10 ml of DMF, in the presence of 10% Pd/C Catalyst was filtered, 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-OBut(IIIb), Rf0,22 (A), AND 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) previously obtained compound (IIIB) was dissolved in 5 ml of DMF. The solution was cooled to OoWith and with stirring was added 24 μl diphenylphosphinite. The reaction mixture was stirred 6 h at ABOUToC and 24 h at 20oWith, DMF was evaporated to 2 ml and precipitated with ethyl acetate. The precipitate was filtered, washed on the filter with ethyl acetate, ether. The precipitate was re-dissolved by heating in 5 ml of DMF and 2 ml of triptoreline and precipitated with ether. Received 103 mg (98% ) of compound (XIII), [a]2D2- 93,90(1, Asón), Rf0,77 (B) OF 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-OBut(XVII).

To a solution of 776 mg (3.0 mmol) HCl H-Phe-OButin 5 ml of DMF was added 0,41 ml (3.0 mmol) Et3N. the precipitation was filtered, the filtrate was cooled to -10o2SO4, water. An ethyl acetate fraction was evaporated, the residue was dissolved in methanol and treated ion exchange resin DOWEX-1 in the form to the disappearance of p-NITROPHENOL according to TLC. The resin was filtered, washed on the filter with methanol, the filtrate was evaporated to 10 ml and subjected to hydrogenation in the presence of 10% Pd/C Catalyst was filtered, the filtrate was evaporated. The resulting dipeptide N-Tle-Phe-OBut(XVII) was subjected to purification on a chromatographic column (Lobar (310 25) LiChroprep Si 60 (40 63 µm) Merck (Germany). Chromatography produced in isocratic mode, as the eluent used system (). The fraction containing the desired product were evaporated and liofilizovane from the aqueous solution. Received 465 mg (47% ) of compound (XVII); so pl. 104-105oC; [a]2D223,5(C 1, DMF), 31o(1, Asón), Rf0,35 (A) 0,73 (D) OF 0.68 (E) 0,80 (W).

Example 25. Z-Ile-Tle-Phe-OBut(XVIII).

To a solution of 435 mg (1.3 mmol) of the compound (XVII) obtained in example 24, in 5 ml of DMF, cooled to 10oC, was added a solution of 502 mg (1,3 which was Adali ether. The precipitate was dissolved in methanol and treated ion exchange resin DOWEX-1 in the form to the disappearance of the ONp according to TLC. The resin was filtered, washed on the filter with methanol, the methanol was evaporated.The resulting oil crystallizable from a mixture methanolate. Received 608 mg (81% ) of compound (XVIII); so pl. 85-86oC; [a]2D2- 18(1, Asón); Rf0,76 (S) 0,90 (W), 0,55 (M).

Example 26. Z-Val-Ile-Tle-Phe-OBut(XIX).

291 mg (0.5 mmol) of the compound (XVIII) obtained in example 25, was first made in 5 ml of methanol in the presence of 10% Pd/C catalyst was filtered, the filtrate was evaporated, the residue was dissolved in 10 ml of DMF and cooled to -10oC. To the resulting solution was added 205 mg (0.55 mmol) Z-Val-ONp in 2 ml of DMF and was kept for 20 h at 20oC. the Solution was evaporated and the oil was dissolved in ether and precipitated with hexane. The precipitate was recrystallized from methanol-water. Received 245 mg (72%) of compound (XIX); so pl. 205-206oC; [a]2D2- 34(1, Asón); Rf0,63 (S) OF 0.85 (W), 0,43 (M).

Example 27. Z-Lys(Boc)-Val-Ile-Tle-Phe-OBut(XX).

204 mg (0.3 mmol) of the compound (XIX) obtained in example 26, was first made in 10 ml of DMF in the presence of 10% Pd/C Catalyst was filtered, filtrative, the residue was dissolved in ether and precipitated with hexane. The procedure was repeated twice. Obtain 125 mg (47% ) of compound (XX); so pl. 229-230oC; [a]2D2- 25(c 1, Asón); Rf0,25 (S) 0,86 (W), 0,78 (A), 0,41 M).

Example 28. Z-Ser-Lys(Boc)-Val-Ile-Tle-Phe-OBut(XXI).

98 mg (0.11 mmol) of the compound (XX) obtained in example 27, was first made in methanol in the presence of 10% Pd/C Catalyst was filtered, the filtrate was evaporated, the residue was dissolved in DMF and cooled to -10oC. To the solution was added 63 mg (0.13 mmol) of Z-Ser-OPcp. Kept for 20 h at 20oWith, DMF was evaporated, the residue was dissolved in a mixture of methanol with ether and precipitated with hexane. The precipitate was separated by filtration, was dissolved in a mixture of chloroform ether hexane and left to crystallize for 20 h at 4oC. the resulting crystalline precipitate was filtered and washed on the filter with hexane. Received 93 mg (85%) of compound (XXI); so PL 231-232oC; [a]2D2- 33(1, Asón); Rf0,70 (A) 0,90 (W). 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).1H NMR spectrum table. 7.

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

85 mg (of 0.085 mmol) of the compound (XXI) obtained in example 28, was first made in DMF in the of Isadora to the solution was added 49 mg (of 0.085 mmol) of the compound (VIII), obtained in example 13. The mixture was cooled to -25oWith and under vigorous stirring was added a solution of 244 mg (0.26 mmol) of the complex F, cooled to the same temperature. The reaction mixture was stirred for 30 min at -25oC, followed by 20 h at 4oC and 24 h at 20oC. the precipitate was Filtered N,N'-dicyclohexylmethane, 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. > 260oC; [a]2D2- 72(c 1, Asón); Rf0,72 (D) 0,51 (E) 0,59 (C).

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

91 mg (0.06 mmol) of the compound (XXII) obtained in example 29 was dissolved in triperoxonane acid and passed a current of dry Nug for 45 min, then the solution was evaporated and the oil triturated with ether, the precipitate was filtered and washed on the filter with ether. The resulting material was subjected to purification by the method of preparative HPLC. Received 60 mg (89%) of compound (XXIII); so pl. 260oC; [a]2D2- 81(1, Asón); Rf0,52 (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).

Ali, the residue was dissolved in 25 ml of chloroform and subjected to chromatographic purification in the same terms as specified in example 21. Obtained 4.3 g (94%) of compound (XXIV); so pl. 131-132oC; [a]2D2- 15,2(C 1, DMF); Rf0,70 (B), AND 0.50 (E) 0,63 ().

Example 32. Z-Leu-Phe-Arg-OH (XXV).

of 4.25 g (9.3 mmol) of the compound (XXIV) obtained in example 31, was first made in 50 ml of methanol in the presence of 10% Pd/C for 2 h, Rf0,38 (B) 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. To a cooled to -10oTo the solution was added 3,86 g (10 mmol) of Z-Leu-ONp. Kept for 30 min at -10oWith and left for 20 h at 20oC. Further processing and chromatographia tions were performed as described in the methodology (XXIV). Received of 4.75 g (90%) of compound (XXV); so pl. 146-147oC [a]2D2- 13,3(1, Asón); Rf0,82 (B) 0,43 (D) OF 0.56 (E).

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

4,55 g (of 8.00 mmol) of the compound (XXV) obtained in example 32, was first made in 100 ml of methanol in the presence of 10% Pd/C, Rf0,50 (B) H-Leu-Ile-Phe-Arg-OH (XXVa). The catalyst was filtered, the filtrate was evaporated. To the residue dissolved in 25 ml of DMF and cooled to -10oYergali chromatographic purification, similar to that described in example 21, with the desired product was elyuirovaniya only when washing the column with a mixture of ethanol, acetic acid, 1:2. The fraction containing the target product was evaporated and the resulting oily residue crystallizable from methanol at 4oC. the resulting crystalline precipitate was filtered, washed on the filter with ether, dried over NaOH in a desiccator. Received 4.35 g (96%) of compound (XXVI); so pl. 189-190oC; [a]2D2- 21,0(1, Asón), Rf0,85 (B) OF 0.45 (D), 0,60 (E).

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

2,05 g (3.00 mmol) of the compound (XXVI) obtained in example 33, was first made in a mixture of DMF N2Oh, 1:1, in the presence of 10% Pd/C Catalyst was filtered, the filtrate was evaporated, the residue was dissolved in 30 ml DMF under heating. To the chilled solution was added a solution of 1.23 g (3,30 mmol of Z-Val-ONp in 5 ml of DMF. The reaction mixture was stirred 48 h at 20oC. To loose amorphous precipitate was added ether and the resulting precipitate was filtered. Twice recrystallized from a mixture of triptoreline methanol, 1:1. Obtained from 2.00 g (85%) of compound (XXVII); so pl. 247-248oC; [a]2D2- 28,5(1, Asón); Rf0,86 (B) OF 0.48 (D), AND 0.62 (E).

Example 35. Z-Lys(Boc)-Val-Ile-Leu-Phe-Arg-OH (XX the accordance 10% Pd/C. The catalyst was filtered off, washed on the filter with acetic acid and the filtrate was evaporated. The residue several times persuasively with water. Crystallizable from methanol at 4oC. was Dried over NaOH in a desiccator. Received of 1.23 g (95%) of H-Val-Ile-Leu-Phe-Arg-OH (XXVIIa), Rf0,48 (B). HPLC: 24,01 min (Syst. 1, deg. 1), 18,07 min (Syst. 2, deg. 1).

0.65 g (1.00 mmol) of the above compound (XXVIIa) was dissolved in a mixture of DMF DMSO, 3: 1, was cooled to -10oWith and added 0,58 g (1.15 mmol) of Z-Lys(Boc)-ONp. Was stirred 48 h at 20oC. the Solvent was evaporated to 3 ml and was added ether. The precipitate was filtered off, washed on the filter with ether. Recrystallized twice from triptoreline. Received 0,57 g (57% ) of compound (XXVIII); so pl. 260oC (decomposition); [a]2D2- 19,0(1, 75% of the Asón); Rf0,88 (B) OF 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 the compound (XXVIII) obtained in example 35, was first made in acetic acid in the presence of 10% Pd/C Catalyst was filtered off, washed on the filter with acetic acid and the filtrate was evaporated and the residue several times persuasively with water. Crystallizable from a mixture of isopropyl alcohol and hexane. The precipitate was dried over NaOH in a vacuum-ExG (of 0.65 mmol) of the above compound (XXVIIIa) was dissolved in a mixture of DMF DMSO, 3:1, was cooled to -10oWith and added to 0.37 g (to 0.72 mmol) of Z-Ser-OPcp. The reaction mixture was stirred 48 h at 20oC. the Solvent was evaporated, the residue when heated was dissolved in 5 ml of DMF and precipitated with ether. The procedure was repeated twice. The precipitate was filtered, washed on the filter with ether. Obtained 0.55 g (77%) of compound (XXIX); [a]2D2- 54,5(1, 50% of the Asón); Rf0,80 (B) 0,59 (E) OF 0.64 (W), 0,70 (K), AND 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 first made in acetic acid in the presence of 10% Pd/C Catalyst was filtered off, washed on the filter with acetic acid and the filtrate was evaporated. The residue several times persuasively with water and n-butanol. Crystallizable out of the water. Retrieved 250 mg (95%) of H-Ser-Lys(Boc)-Val-Ile- -Leu-Phe-Arg-OH (XXIXa), Rf0,48 (B), HPLC: 33,81 min (Syst. 1, deg.1). 27,46 min (Syst. 2, deg.1)1H NMR spectrum table. 8.

54 mg (0,094 mmol) of the compound (VIII) obtained in example 13, was dissolved in 2 ml of DMF, cooled to -25oWith and with stirring was added 270 mg (0,282 mmol) of the complex F in 1.5 ml DMF. Was stirred for 30 min at -25oC, 24 h at 2oC. Officine 73 mg (96%) of Z-Glp-Pro-Pro-Gly-Gly-Opcp, Rf0,85 (B) who immediately after precipitation was dissolved in 2 ml of DMF, cooled to -25oWith and added with stirring to a solution of 85 mg (0.90 mmol) previously obtained compound (XXIXa) in a mixture of 3 ml of DMF and 4 ml hexamethylphosphoramide (Fluka). Was stirred for 30 min at -25oC, 72 h at 20oC. DMF was evaporated, the residue is precipitated with ether. Received 128 mg (94%) of compound (XXX), [a]2D2- 94,0(1, Asón), Rf0,73 (B) 0,35 (E) 0,75 (O) OF 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 triperoxonane acid and passed a current of dry Nug for 1 h, the Solution was evaporated, the residue triturated with ether and was 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]2D2- an 81.25(1, Asón), Rf0,40 (B), 0,16 (E) 0,80 (N) AND 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),1H NMR spectrum table. 9.

Example 39. Z3-Arg-Val-Ile-Leu-Phe-Arg-OH (XXXII).

To a suspension of 253 mg (0.4 mmol) of the compound (XXVIIa), p is ing a mixture of 48 h at 20oC. To loose amorphous precipitate was added ether and was filtered. Several times perioadele from DMF-ether. Received 268 mg (56%) of compound (XXXII); so pl. 242-244oC; [a]2D2- 24,3(1, Asón); Rf0,90 (B), AND 0.50 (D) 0,55 (E).

Example 40. Z-Ser(But)-Arg-Val-Ile-Leu-Phe-Arg-OH (XXXIII).

241 mg (0.20 mmol) of the compound (XXXII) obtained in example 39, was first made in acetic acid in the presence of 10% Pd/C for 3 hours, the Catalyst was filtered, the filtrate was evaporated and the residue was besieged from methanol-ether. The precipitate was dried in a vacuum desiccator over NaOH. Received 157 mg (98%) H-Arg-Val-Ile-Leu-Phe-Arg-OH (XXXIIa), Rf0,15 (B). HPLC: 23,26 min (Syst. 1, deg. 1), 16,00 min (Syst. 2, deg.1).

145 mg (0.18 mmol) of the compound (XXXIIa) was dissolved in a mixture of DMF DMSO, 2: 1, was cooled to -10oWith and added 99 mg (0.22 mmol) of Z-Ser(But)-ONB. Was stirred 48 h at 20oC. the Solution was evaporated at 90% volume and precipitated with ether. The precipitate was filtered, washed on the filter with ether, dried and dissolved when heated in 3 ml of DMF. Precipitated upon cooling the amorphous precipitate was filtered, washed on the filter is cooled DMF, ether, and dried. The procedure was repeated twice. Obtained 130 mg (67%) of compound (XXXIII); so pl. 253-256oC (with decomposition(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 the compound (XXXIII) obtained in example 40, was first made in acetic acid in the presence of 10% Pd/C and was isolated in conditions similar to those specified in example 40. Received 95 mg (100%) of H-Ser(But)-Arg-Val-Ile-Leu-Phe-Arg-OH (XXXIIIa), Rf0,67 (C), 0,48 (K) 0,30 (D).

44 mg of Glp-Pro-Pro-Gly-Gly-OH was dissolved in 15 ml of DMF, cooled to -25oWith and with stirring was added a solution of the complex F in 5 ml of DMF. The precipitation was filtered N N dicyclohexylamine, the solvent was evaporated to 1/3 volume and precipitated with ether. The precipitate was filtered and dissolved in 2 ml of DMF. The resulting solution was cooled to -10oWith and added to a solution of 95 mg of compound (XXXIIIa) in 20 ml of DMF. To the solution was added to 13.5 mg (0.1 mmol) t. Was stirred 48 h at 20oC. the Solvent was evaporated, the residue was dissolved in methanol and precipitated with ethyl acetate. Received 90 mg (70%) of compound (XXXIV), Rf0,45 (C).

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

90 mg (0.07 mmol) of the compound (XXXIV) obtained in example 41 was dissolved in triperoxonane acid and kept for 1 h at 20oC. Triperoxonane acid was evaporated, the residue triturated with ether is I (XXXV), [a]2D2- 60(c 1, Asón); Rf0,43 (B), 0,32 (H), 0.75 IN (H) OF 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), received 19.82 min (Syst. 2, deg. 1).1H NMR spectrum table. 10.

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

Was obtained from HPLC-purification obtained in example 42, the connection in the amount of 10% of the initial number of connections (XXXIIIa) is similar to that given in example 41. Received 9 mg (Hjhb), [a]2D2- 26(1, Asón), Rf0,38 (B) OF 0.75 (H). HPLC: 23,15 min (Syst.1, deg. 1). 16,59 min (Syst.2B deg.1),1H NMR spectrum table. 11.

Sources of information taken into account:

1. Birr C. Zachmann B. Bodenmuller, H. Shcaller H. C. "Synthesis of a new neuropeptide, the head activator from hydra", Febs. Lett. 1981, V. 131, No. 1, p. 317-321.

1. Derivatives of the peptides of General formula I

A Ile Y Phe B,

where a N, H - Val -, H Ser Val X -, Glp Pro Pro Gly Gly - Ser-X-Val -;

B-OH, -Arg-OH, X-LyS -, -Arg -;

Y Leu -, -Tle,

where Tle L-tert-leucine.

2. The derived peptide under item 1, representing undecapeptide following amino acid sequence

Glp Pro Pro Gly), Ser Arg Val Ile Leu Phe OH.

3. The derived peptide under item 1, representing hundecape peptide under item 1, representing a peptide of the following amino acid sequence

Glp Pro Pro Gly Gly Ser Lys Val Ile Tle Phe-Arg - OH.

5. The derived peptide under item 1, which represents a peptide of the following amino acid sequence

Glp Pro Pro Gly)2Ser Arg Val Ile Tle Phe-Arg-OH.

6. The method of obtaining derivatives of the peptides of General formula I

A Ile Y Phe B,

where A H-, H-Val -, H Ser Val X -, Glp Pro Pro Gly Gly - Ser-X-Val -;

B OH, Arg-OH, X-Lys -, -Arg -;

Y Leu -, Tle,

characterized in that perform sequential build-up of the peptide chain from the C-end amino acid sequence with the activated ester protected at the alpha-amino group of amino acids and any resulting compound of the formula A Ile Y Phe B,

where A: H-, H-Val -, H-Ser-X-Val -,

values, X and Y mentioned above,

if necessary, in the case where a N Ser Val X-expose fragment condensation in the presence of dicyclohexylcarbodiimide and additives, excluding racemization, with the corresponding N-terminal peptide to obtain compound I

where A H - Glp Pro Pro Gly Gly Ser Val X.

7. The method of obtaining derivatives of the peptides of General formula I

A Jle Leu Phe OH, where A HGlp-Pro-Pro-Gly-Gly - Ser-Arg-Val-,

characterized in that the consistently increasing Patinoire of tetrapeptide Val - Ile - Leu, Phe, and carry out the condensation of the resulting peptide fragments by sequential accession to the above-mentioned N-terminal Pentapeptide of the dipeptide Ser Arg using dicyclohexylcarbodiimide in the presence of additives, excluding racemization, and by joining the N-terminal heptapeptide Glp Pro Pro Gly Gly Ser Arg C-terminal tetrapeptide Val Ile Leu Phe using diphenylphosphinite.

 

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