RU2756772C2 - Tspo dipeptide ligands having neuropsychotropic activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to new TSPO dipeptide ligands having the range of neuropsychotropic activity. Dipeptide of the general formula R₁-X-AК₁-Y-AК₂-R₂ is proposed, where X=L or D configuration, Y=L or D configuration, АК₁=Trp or Gly, АК₂=Leu, R₁=C₆H₅-(CH₂)₂-C(O), С₆Н₅-СН₂-С(O) or CH₃-(CH₂)₄C(O), R₂=OH, OCH₃, NH₂ or NHCH₃. A pharmaceutical composition of the specified dipeptide having neuropsychotropic activity, a method for the treatment of anxiety states, and a method for the treatment of depressive states by injecting effective amount of the specified dipeptide are also proposed. Anxiolytic activity of dipeptides was identified in doses of 0.1-1.0 mg/kg, and for C₆H₅-(CH₂)₂C(O)-L-Trp-L-Leu-NH₂ – in doses of 0.01-5.00 mg/kg. Antidepressant activity for C₆H₅-(CH₂)₂C(O)-L-Trp-L-Leu-NH₂ was identified in doses of 0.01-0.05 mg/kg.

EFFECT: obtaining TSPO dipeptide ligands with neuropsychotropic activity.

13 cl, 1 dwg, 14 tbl, 10 ex

Description

Scope of invention

The invention relates to new biologically active dipeptides
general formula:

where AK ₁ = Tgr or Gly, AK ₂ = Leu;

X = L or D configuration, Y = L or D configuration;

R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O) or C ₆ H ₅ -CH ₂ -C (O) or CH ₃ - (CH ₂ ) ₄ -C (O);

R ₂ = OH or OCH ₃ or NH ₂ or NHCH ₃ ,

The new compounds are ligands of a translocator protein (18 kDa, TSPO) and have neuropsychotropic properties, in particular, anxiolytic and antidepressant properties.

State of the art

Translocator protein (TSPO) discovered in 1977 [Braestrup C, Squires R.F., Proc. Natl. Acad. Sci. USA. Sep 1977; 74 (9): 3805-3809.] Is a transmembrane polypeptide consisting, depending on the species, of 153-169 amino acids, localized mainly on the outer mitochondrial membrane and is responsible for the transfer of cholesterol from the outer mitochondrial membrane to the inner one. In addition, TSPO is involved in porphyrin transport, mitochondrial respiration, mitochondrial pore opening, apoptosis, and cell proliferation. Until 2006, TSPO was known as a peripheral benzodiazepine receptor (PBR), however, due to a better understanding of the mechanism of action, it was renamed to translocator protein (TSPO) [Papadopolous V., Baraldi M., Guilarte TR, Knudsen TB, Lacapère J.-J., Lindemann P. et al. Translocator protein (18 kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol. Sci. 2006; 27, no. 8: 402-409]. The translocator protein is located on the outer mitochondrial membrane mainly in steroid-generating cells, but is also present in other tissues and organs, such as kidneys, lungs, heart, and is also presented in glial cells of the brain [Beurdeley-Thomas A., Miccoli L., Oudard S., Dutrillaux B., Poupon MF, J. Neurooncol. 2000. V. 46. No. 1. P. 45-56. Also, a high content of TSPO was found in some types of neuronal cells, such as neurons of the mammalian olfactory bulb [Anholt R.R., Murphy K.M., Mack G.E., Snyder S.H. Peripheral-type benzodiazepine receptors in the central nervous system: localization to olfactory nerves. J. Neurosci. 1984 .; 4: 593-603, Bolger G.T., Mezey E., Cott J., Weissman B.A., Paul S.M., Skolnick P. Differential regulation of 'central' and 'peripheral' benzodiazepine binding sites in the rat olfactory bulb. Eur. J. Pharmacol. 1984; 105: 143-148].

The main endogenous ligands are cholesterol and porphyrins; this group also includes endosepins, which are neuropeptides. Cholesterol is a key endogenous ligand of TSPO, since it is he who is the starting substance for further biosynthesis of neurosteroids after penetrating the mitochondrial membrane. Cholesterol has a nanomolar affinity for TSPO (Kd <10 nM). By binding to TSPO, cholesterol is transferred from the outer membrane of mitochondria to the inner membrane, where, under the action of cytochrome P450scc enzymes, 3-β hydroxysteroid dehydrogenase (3β-HSD), 5α-reductase, 3-α hydroxysteroid dehydrogenase (3α-HSD), a cascade , which perform the modulatory function of GABA _A. In terms of their strength, they are either equal to or superior to benzodiazepines and barbiturates.

A number of porphyrins are also considered endogenous TSPO ligands. The following porphyrin derivatives have the highest affinity: protoporphyrin IX (Ki = 14.5 ± 10.7 nM), deuteroporphyrin IX (Ki = 31.3 ± 2 nM), and hemin (Ki = 40.6 ± 13.7 nM). Despite the fact that many porphyrins have high affinity for TSPO, these compounds have hardly been studied for the presence of biological effects. During the search for endogenous TSPO ligands, it was found that one of them is the diazepam binding inhibitor (DBI). DBI is a 9 kDa polypeptide that was discovered in 1983 in the rat brain while studying proteins that inhibit benzodiazepine binding to the GABA _A receptor. It has also been shown that the main post-translational products of DBI, octadecaneuropeptide DBI _33-50 (octadecaneuropeptide, ODN) and triakontatetroneuropeptide DBI _17-50 (triakontatetraneuropeptide, TTN), also have an affinity for TSPO.

Diazepam, clonazepam, and some other benzodiazepine derivatives have long been considered synthetic ligands of TSPO, but these substances bind to both TSPO and the central benzodiazepine receptor.

The first synthetic TSPO ligands that were used for in vivo studies are: isoquinoline derivative PK-11195, benzodiazepine derivative Ro5-4864.

The authors who first published data on PK11195 did not present in their work an approach to the design of this molecule, on the basis of which it can be assumed that it was discovered by wide screening [Le Fur G., Perrier ML, Vaucher N., Imbault F., Flamier A., Benavides J. et al. Peripheral benzodiazepine binding sites: effect of PK 11195, 1- (2-chlorophenyl) -N-methyl-N- (1-methylpropyl) -3-isoquinolinecarboxamide. I. In vitro studies. Life Sci. 1983; eighteen; 32 (16): 1839-47]. The study of the effect of PK-11195 on neurosteroidogenesis showed that with the simultaneous administration of PK-11195 and Ro5-4864, the effect of the latter decreased. Based on the fact that Ro5-4864 is an agonist, it was concluded that PK-11195 is a TSPO antagonist. PK11195 has been widely used since its discovery until now as the "gold standard" of TSPO ligands in studies related to this receptor.

A number of pharmaceutical companies are intensively searching for ligands - TSPO agonists in order to create drugs for the treatment of neurological and mental diseases. So, in the company Dainippon Sumitomo Pharma (Japan) together with the company Novartis Pharmaceuticals (USA), the TSPO ligand AC-5216 (XBD173, Emapunil) was created, [Kita A. et al. Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand // British journal of pharmacology. - 2004. - T. 142. - No. 7. - C. 1059-1072], a derivative of oxopurine, which showed anxiolytic and antidepressant effect in the Vogel tests, in the test there is a dark-light chamber. AC-5216 (XBD173, Emapunil) has passed the II stage of clinical trials as an anxiolytic with an antidepressant component of action. To investigate the anxiolytic potential, XBD173 was tested in healthy male volunteers using the cholecystokinin tetrapeptide (CCK4). CCK-4 is a fragment of the cholecystokinin hormone known for its anxiety and gastrointestinal effects, but less pronounced than other forms of cholecystokinin. In humans, CCK-4 causes a marked sense of anxiety, starting at doses of 50 mg, and therefore the drug is used to induce panic attacks and to test possible anxiolytics. A Phase II clinical trial on the efficacy, safety and tolerability of XBD173 was conducted by Novartis in patients with anxiety disorders. [Efficacy, safety and tolerability of XBD173 in patients with generalized anxiety disorder "ClinicalTrials.gov Identifier: NCT00108836]

The authors of the article [Campiani, G .; Nacci, V .; Fiorini, L .; De Filippis, M. P .; Garofalo, A .; Ciani, S.M .; Greco, G .; Novellino, E .; Williams, D. C .; Zisterer, D.M .; Woods, M. J .; Mihai, C; Manzoni, C; Mennini, T.J. Med. Chem., 1996, 39, 3435-50] studied ligands of the benzoxyazepine class under conditions of competitive displacement PK-11195. Compound OXA-17f was noted as the most selective of this series (Ki = 0.26 cM).

The general formula of the TSPO ligands of the benzoxyazepine series (OXA)

OXA-17f connection

In 1990 by the authors [Nacci, V .; Fiorini, I .; Garofalo, A .; Cagnotto, A. Farmaco, 1990, 45, 545-557.] A new class of TSPO benzothioazepine ligands has been proposed. Some of these ligands, such as THIA-66 and THIA-67, have high affinity and selectivity for TSPO, however, it has been proven that they bind to TSPO less strongly than PK-11195 [Fiorini I., Nacci V., Ciani SM, Garofalo A., Campiani G., Savini L., Novellino E., Greco G., Bernasconi P., Mennini TJ A comparative molecular field analysis model for 6-arylpyrrolo [2,1-d] [1,5] benzothiazepines binding selectively to the mitochondrial benzodiazepine receptor. // J Med Chem. - 1994. - No. 37. - P. 4100-4108]. These ligands are useful as probes in in vitro radio studies.

THIA-66 and THIA-67 connections

The article (Romeo, EA. Et al. J. Pharmacol. Exp. Ther., 1992, 262, 971-978) describes arylindoleacetamide derivatives showing TSPO agonist activity. Arylindoleacetamide SSR180575 [Ferzaz B., Brault E., Bourliaud G., Robert JP, Poughon G., Claustre Y., Marguet F., Liere P., Schumacher M., Nowicki JP, Fournier J., Marabout B. , Sevrin M., George P., Soubrie P., Benavides J., Scatton B. SSR180575 (7-chloro-N, N, 5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H- pyridazino [4,5-b] indole-1-acetamide), a peripheral benzodiazepine receptor ligand, promotes neuronal survival and repair. J Pharmacol Exp Ther. 2002 Jun; 301 (3): 1067-78] has a nanomolar affinity for TSPO four times that of Ro-5 4864 in both rats and humans. SSR180575 increases pregnenolone levels in the brain, which means that its therapeutic effects are mediated through its ability to stimulate steroidogenesis. SSR180575 is in a second phase of clinical trials (Sanofi-Aventis, NCT00502515) for the treatment of diabetic neuropathy.

Connection SSR180575

In 1991, Synthelabo (now Sanofi-Aventis) considered the imidazopyridine derivative Alpidem as a potential TSPO ligand. However, in 1995, Alpidem was discontinued after the discovery of liver dysfunction caused by it, with serious consequences and even death. Also, these side effects may be associated with its binding to GABA receptors, which can sometimes also be found in liver tissue - for example, in hepatocellular carcinoma cells. In 1997 the authors of the article [Trapani, G .; Franco, M .; Ricciardi, L .; Latrofa, A .; Genchi, G .; Sanna, E .; Tuveri, F .; Cagetti, E .; Biggio, G .; Liso, G.J. Med. Chem., 1997, 40, 3109-18] conducted a number of studies in an attempt to develop a new spectrum of imidazopyridine derivatives with improved selectivity towards TSPO. The compound CB-34, developed by them, turned out to be the most promising due to its high affinity, selectivity and ability to stimulate steroidogenesis in peripheral tissues.

Compound SV-34

Phenoxyphenylacetamide derivatives, N- (4-chloro-2-phenoxyphenyl) -N- (2-isopropoxybenzyl) acetamide, DAA1097 and N- (2,5-dimethoxybenzyl) -N- (5-fluoro-2-phenoxyphenyl) acetamide, DAA1106 are considered TSPO ligands. These compounds exhibit potent anxiolytic effects in in vivo experiments. [Chaki S., Funakoshi T., Yoshikawa R., Okuyama S., Okubo T., Nakazato A., Nagamine M., Tomisawa K. Neuropharmacological profile of peripheral benzodiazepine receptor agonists, DAA1097 and DAA1106. // Eur. J. Pharmacol. - 1999. - No. 371. - P. 197-204]

Connections DAA1097 and DAA1106

The authors of the RF patent 2573823 (priority date 03/26/2014) disclose the world's first dipeptide TSPO ligands of the general formula R ₁ -C (O) -X-Trp-Y-Ile-R ₂ possessing anxiolytic properties, where X = L or D configuration, Y = L configuration, R ₁ = C ₆ H ₅ -CH ₂ -O- or C ₆ H ₅ - (CH ₂ ) ₅ - or C ₆ H ₅ -CH ₂ -, R ₂ = OH or OCH ₃ or NH ₂ or NHCH ₃ .

As an example of TSPO ligands with anxiolytic and antidepressant activities, there are a number of pyrrolo [1,2-α] pyrazine derivatives disclosed in RF patent 2572076.

However, the compounds described in the above information do not disclose or suggest new structural variations of the patented compounds.

The relevance of the problem solved with the help of this invention

Epidemiological data indicate that anxiety states, including generalized anxiety, phobic, panic and obsessive-compulsive disorders according to ICD-10, occur in 6-10% of the population. Anxiety conditions, comorbid with somatic diseases, aggravate the latter, increasing, for example, mortality in cardiovascular diseases by half. Current drugs for the treatment of anxiety disorders are not effective enough. Fast-acting benzodiazepine tranquilizers cause sedation, muscle relaxation, addiction and dependence. For serotonergic anxiolytics, free from these side effects, a two-week period of onset of effect is required. As a result, it is urgent to create a drug aimed at regulating the neurosteroid mechanisms of cytoprotection, combining the properties of a fast-acting anxiolytic and antidepressant. A promising approach to the creation of new drugs with a wide spectrum of neuropsychotropic activity, including anxiolytic, antidepressant, and nootropic, is to obtain new ligands of the TSPO translocator protein. Since TSPO plays a critical role in the regulation of neurosteroid synthesis, the ligands of this receptor can be effective neuropsychotropic drugs, for example, antidepressants, anxiolytics, nootropics, or complex drugs. The original TSPO ligands are supposed to be created on the basis of substituted tryptophanilleucyl-containing dipeptides.

The essence of the invention

Our goal was to obtain TSPO dipeptide ligands with a spectrum of neuropsychotropic activity, in particular anxiolytic and antidepressant. We have found that this goal can be achieved using compounds of the general formula:

R ₁ -X-AK ₁ -Y-AK ₂ -R ₂

where AK ₁ = Tgr or Gly, AK ₂ = Leu; X = L or D configuration, Y = L or D configuration; R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O) or C ₆ H ₅ -CH ₂ -C (O) or CH3- (CH ₂ ) ₄ -C (O); R ₂ = OH or OCH ₃ or NH ₂ or NHCH ₃ .

The following compounds can serve as examples of the invention:

I. N-phenylpropionyl-L-tryptophanyl-L-leucine amide

II. N-phenylpropionyl-D-tryptophanyl-L-leucine amide

III. N-phenylpropionyl-L-tryptophanyl-D-leucine amide

IV. N-phenylpropionyl-L-tryptophanyl-L-leucine methyl ester

V. N-phenylpropionyl-L-tryptophanyl-L-leucine

Vi. N-phenylpropionyl-L-tryptophanyl-L-leucine methylamide

Vii. N-phenylacetyl-L-tryptophanyl-L-leucine amide

VIII. N-caproyl-L-tryptophanyl-L-leucine amide

IX. N-phenylpropionyl-glycyl-L-leucine amide

X. N-phenylpropionyl-L-tryptophanyl-glycine amide

The compounds provided in the invention are additionally illustrated in table. 1.

The above compounds were prepared by well known peptide synthesis methods. The usual process for the preparation of the subject compounds consists in mixing and condensation of the desired amino acids, usually in a homogeneous phase.

Homogeneous phase condensation can be performed as follows:

a) condensing an amino acid having a free carboxyl group and a protected other reactive group with an amino acid having a free amino group and protected other reactive groups in the presence of a condensing agent such as a carbodiimide;

b) condensation of an amino acid having an activated carboxyl group and a protected other reactive group with an amino acid having a free amino group and protected other reactive groups;

c) condensation of an amino acid having a free carboxyl group and a protected other reactive group with an amino acid having an activated amino group and protected other reactive groups.

The carboxyl group can be activated by converting it to a chloride, azide, anhydride group, or an activated ester such as N-hydroxysuccinimide, N-hydroxybenzotriazole, pentachlorophenyl or paranitrophenyl esters. The amino group can be activated by converting it to phosphitamide or by the "phosphorase" method.

The most common for the condensation reactions discussed above are: carbodiimide method; azide method; mixed anhydride method; method of activated ethers. These methods are described in "The Peptides". Vol. 1. 1965 (Academic Press), E. Schroeder, K. Lubke, or in "The Peptides", Vol. 1, 1979 (Academic Press) E. Cross, L. Meinhofen.

Preferred condensation methods for the preparation of peptides of formula (1) is the activated ester method, which is preferably carried out using amino acid protected succinimide esters. The best solvents are a mixture of ethyl acetate with dichloromethane, pure ethyl acetate and tetrahydrofuran.

Reactive groups that should not participate in condensation can be protected with groups that are easily removed, for example, by hydrolysis or reduction. Thus, the carboxyl group can be protected by etherification with methanol, ethanol, tert-butanol, benzyl alcohol.

The amino group is usually effectively protected with acidic groups, for example, aliphatic, aromatic, heterocyclic carboxylic acid residues such as acetyl, benzoyl, pyridinecarboxyl; acidic groups derived from carbonic acid such as ethoxycarbonyl, benzyloxycarbonyl, tert-butyloxycarbonyl; or acidic groups derived from sulfonic acids such as p-toluosulfonyl.

The protecting groups were removed in accordance with the nature of these groups. The carbobenzoxy group was removed by catalytic hydrogenolysis in a stream of hydrogen in methanol with the addition of 10% palladium on carbon; The tert-butyloxycarbonyl group was removed in the presence of anhydrous trifluoroacetic acid in dichloromethane.

In the synthesis of the claimed compounds, N-acyl derivatives were obtained using activated succinimide esters of phenylacetic, phenylpropionic and hexanoic acids.

The amides of the dipeptides of formula (1) can be prepared by introducing an amide group into the corresponding dipeptide by any suitable method, for example, by treating the dipeptide with ammonia in the presence of a condensing agent. Alkylamides of peptides are prepared by aminolysis of an alkyl ester of the corresponding dipeptide or by reaction with an amino acid in the form of the desired alkylamide. Esters of peptides according to formula (1) are preferably obtained by using the amino acid in the form of the desired ester. They can also be obtained by appropriate esterification of the obtained peptide. Methyl and ethyl esters are preferred. Compounds with an open carboxyl group are obtained by alkaline hydrolysis of the corresponding dipeptide ester.

By administering effective amounts of a dipeptide of general formula I according to the present invention, a method for treating anxiety and depressive conditions can be realized.

The dipeptide of general formula I, according to the present invention, can, as an active ingredient in a therapeutically effective amount, be included in a pharmaceutical composition having a neuropsychotropic effect.

Examples of implementation of the invention

In what follows, the following abbreviations are used:

Boc - tert-butyloxycarbonyl

DIPEA - diisopropylethylamine

DMAPA - dimethylpropylene diamine

DMSO - dimethyl sulfoxide

Gly - glycyl

Leu - leucyl

Me - methyl

OSu - hydroxysuccinyl

Ph - phenyl

TFA - trifluoroacetic acid

Tgr - tryptophanil

Pd / C - catalyst: palladium nanoparticles on the surface of activated carbon

Z - benzyloxycarbonyl

DMF - dimethylformamide

THF - tetrahydrofuran

TLC - thin layer chromatography

EA - ethyl acetate

NMR - nuclear magnetic resonance

The following equipment was used to determine the physicochemical characteristics of the obtained substances:

- melting points were determined on an Optimelt MPA100 instrument (Stanford Research Systems, England) in open capillaries without correction;

- the specific rotation of the plane of polarization of light was recorded on an automatic polarimeter ADP 440 Polarimeter (Bellingham + Stanley Ltd., England);

- ¹ H-NMR spectra were recorded in the δ, ppm scale, on a Fourier-300 spectrometer (Bruker, Germany) in DMSO-d ₆ solutions, using tetramethylsilane as an internal standard. The following abbreviations were used to designate the resonance signals: s - singlet, d - doublet, d d - doublet of doublets, t - triplet, m - multiplet;

- thin layer chromatography (TLC) was performed on silica gel plates DC Kieselgel 60 G / F ₂₅₄ (Merck, Germany) in solvent systems chloroform: methanol, 9: 1 (A) or isopropyl alcohol: aqueous ammonia, 7: 3 (B). Compounds containing amide groups were identified by a chloro-tolidine test; compounds containing aromatic groups - in ultraviolet rays, and compounds containing open carboxyl groups and ester groups - 5% alcohol solution of bromcresol green

EXAMPLE 1. Obtaining amide N-phenylpropionyl-L-tryptophanyl-L-isoleucine (I), Ph (CH ₂ ) ₂ -C (O) -L-Trp-L-Ile-NH ₂ .

a) N-tert-butyloxycarbonyl-L-leucine, N-Boc-L-Leu-OH.

Dissolved 8.00 g (60.98 mmol) of L-leucine in a mixture of 50 ml of 1N NaOH, 100 ml of water, 200 ml of isopropyl alcohol. After complete dissolution, 15.97 g (73.17 mmol, 20% excess) of di-tert-butylpyrocarbonate were added. The mixture was stirred for 2 h at room temperature, maintaining pH = 9-10 by dropwise addition of a 1N NaOH solution. The progress of the reaction was monitored by TLC in system A. After the completion of the reaction, the reaction mass was evaporated in a vacuum rotary evaporator to one third of the volume, while isopropyl alcohol was mainly removed. Excess di-tert-butylpyrocarbonate was extracted with hexane (2 × 100 ml). The aqueous solution was acidified with 10% H ₂ SO ₄ solution to pH 4, the product precipitated as a white "curd" precipitate. The precipitate was allowed to stand for 12 h, then filtered off, washed with distilled water until neutral, and dried in air in a porcelain cup to constant weight. Received 11.14 g (79%) of the product in the form of a white powder with so pl. 82-83 ° and [α] _D ²⁰ -23 ° (c = 1, acetic acid), (lit. data 83 ° C [α] _D ²⁰ -24 ° (c = 1, acetic acid [A.A. Gershkovich , VK Kibirev. Chemical synthesis of peptides. Kiev: Naukova Dumka, 1992]).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.85 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.08-1.22 (1 H , m, C ^γ H ₂ Leu), 1.36 (9 H, s, -OC (CH ₃ ) ₃ ), 1.45 (2 H, m, C ^β H Leu), 3.86-3.88 (1 H, dd, C ^α H Leu), 6.72 (1 H, d, NH Leu), 6.89 and 7.19 (2 H, two s, NH ₂ amide).

b) N-tert-butyloxycarbonyl-L-leucine succinimide ester, N-Boc-L-Leu-OSu.

To a solution of 8.00 g (34.63 mmol) of Boc-L-leucine in 150 ml of THF was added 4.73 g (39.88 mmol, 15% excess) of N-hydroxysuccinimide, the solution was cooled to + 5 ° C in a water bath with ice, then 8.26 g (39.88 mmol, 15% excess) of dicylohexylcarbodiimide was added. The reaction mixture was stirred for 30 min at + 5 ° С and for 5 h at room temperature. The progress of the reaction was monitored by TLC in system A. At the end of the reaction, the precipitate of dicyclohexylurea was filtered off, the filtrate was evaporated. The resulting viscous oil was dissolved in 30 ml of dichloromethane. The solution was kept for 24 h in a refrigerator at + 8 ° С, the re-formed precipitate of dicyclohexylurea was filtered off, the organic phase was washed with 5% NaHCO ₃ (2 × 100 ml) and 100 ml of distilled water. It was dried over Na ₂ SO ₄ , the desiccant was filtered off, the filtrate was evaporated, and a thick white oil was obtained, which was crystallized in isopropyl alcohol. The crystals obtained were kept for 12 h in a refrigerator at + 8 ° С, and the product was obtained in the amount of 8.31 g (73%) in the form of glassy crystals, mp. 110-112 ° C, [α] _D ²⁶ = -40 ° (c = 2, dioxane)). (Ref. Data: mp 116 ° C (crystal from diisopropyl ether), [α] _D ²⁵ = -41.8 ° (c = 2, dioxane) [Anderson, GW; Zimmerman, JE; Callahan, FM The use of esters of N-hydroxysuccinimide in peptide synthesis J. Am. Chem. Soc. 1964, 86, 1839-1842]).

¹ H-NMR (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.39 (9 H, s, -ОС (CH ₃ ) ₃ ), 1.45 (2 H, t, C ^β H Leu), 1.56-1.73 (1 N, m, C ^γ H ₂ Leu), 2.82 (4 N, m , OSu), 3.86-3.88 (1 H, m, C ^α H Leu), 7.93 (1 H, d, NH Leu).

c) N-tert-butyloxycarbonyl-L-leucine amide, N-Boc-L-Leu-NH ₂ .

To a solution of 8.00 g (24.36 mmol) Boc-L-Leu-OSu in 15 ml of DMF was added 100 ml of an aqueous ammonia solution with stirring; a white curdled precipitate immediately formed. The resulting suspension was stirred for 30 min until the reaction was complete. The precipitate was allowed to stand for 3 h in a cool place, but not in a refrigerator, then the formed precipitate was filtered off on a glass filter, washed with distilled water until the wash water was neutral, and dried in an oven at 100 ° C until constant weight. Received 3.37 g (60%) Boc-L-Leu-NH ₂ in the form of a white powder with so pl. 148-149 ° C, [α] _D ²⁶ -11.0 ° (c = 1, DMF). (Ref. Data: mp 150-152 ° C, [α] _D ²⁴ -11.9 ° (c = 1, methanol) [C. Somlai G. Szokan, L. Balaspiri. Efficient, racemization-free amidation of protected amino acids Synthesis; 3; (1992); p. 285-287])

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.85 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.08-1.22 (1 H , m, C ^γ H ₂ Leu), 1.36 (9 H, s, -OC (CH ₃ ) ₃ ), 1.45 (2 H, m, C ^β H Leu), 3.86-3.88 (1 H, dd, C ^α H Leu), 6.72 (1 H, d, NH Leu), 6.89 and 7.19 (2 H, two s, NH ₂ amide).

d) L-leucine amide trifluoroacetate, TFA * L-Leu-NH ₂

To a suspension of 12.00 g (52.10 mmol) of Boc-L-Leu-NH ₂ in 30 ml of dichloromethane was added 60 ml of TFA (150% excess) with stirring on a magnetic stirrer, stirred for 2 h. The solution was re-evaporated with diethyl ether (3 × 50 ml) ... The resulting white oil was triturated under diethyl ether to obtain a white powder, the ether was decanted, the residue was filtered off. The product was air dried. Received 11.88 g (99%) TFA * H-Leu-NH ₂ in the form of a white powder with so pl. 118-119 ° C, [α] _D ²⁵ -18 ° (c = 1, DMF).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.83-0.89 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.08-1.22 (1 H , m, C ^γ H ₂ Leu), 1.36 (9 H, s, -OC (CH ₃ ) ₃ ), 1.45 (2 H, m, C ^β H Leu), 3.69 (1 N, d d , C ^α H Leu), 7.52 and 7.93 (2 H, two s, NH ₂ amide), 8.12 (3 H, br.s, N ⁺ H ₃ Leu).

e) N-Carbobenzoxy-L-tryptophan (Z-L-TrpOH).

10.00 g (48.9 mmol) of L-tryptophan was dissolved in 200 ml of 0.1 N NaOH solution. On cooling, a 50% solution of carbobenzoxychloride in toluene (10 ml, 10% excess) and 100 ml of 1N NaOH solution were added dropwise from two dropping funnels. The reaction mass was allowed to warm to room temperature, stirred for 12 h. After the completion of the reaction (TLC control in system B), the alkaline solution was washed with diethyl ether, the aqueous fraction was neutralized with 1 N HCl solution. The precipitate that formed was filtered off, washed with 0.1 N HCl, distilled water, and dried in a vacuum in a desiccator over CaCl ₂ . Received 15.41 g (93%) of the product in the form of a gray powder with so pl. 125-127 ° C. (lit. data 126 ° C [AA Gershkovich, VK Kibirev. Chemical synthesis of peptides. Kiev: Naukova Dumka, 1992]).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 2.98 and 3.18 (2 H, two dd, C ^β H Trp), 4.21 (1 H, m, C ^α H Trp), 5.96 (2 H, s, -OC H ₂ C ₆ H ₅ ), 6.87-7.76 (10 H, m, -OCH ₂ C ₆ H ₅ , indole), 7.45 (1 H, d, NH Trp), 10.80 (1 H, s, NH indol).

f) N-carbobenzoxy-L-tryptophan succinimide ester, Z-L-Trp-OSu.

To a solution of 10.00 g (29.6 mmol) ZL-TrpOH in 300 ml of ethyl acetate was added 3.98 g (34.6 mmol, 17% excess) of N-hydroxysuccinimide, the solution was cooled to + 5 ° С, and then 7.25 (35.2 mmol, 19% excess ) dicylohexylcarbodiimide, making sure that the temperature does not exceed + 5 ° C. The reaction mixture was stirred for 30 min at a temperature of +5 - + 7 ° C and then for 12 h at room temperature. The dicyclohexylurea precipitate was filtered off, the filtrate was evaporated. The resulting viscous oil was dissolved in 200 ml of dichloromethane. The solution was kept for 24 h in a refrigerator at + 8 ° С, the re-formed precipitate of dicyclohexylurea was filtered off, and the filtrate was evaporated. The resulting oil was washed with hexane, the hexane was decanted, the oil was evaporated to dryness. The product was obtained in the amount of 11.84 g (92%) in the form of a white foam, which was crushed to a powder with a so pl. 137-140 ° C [α] ²⁵ _D -60.0 ° (s 1, DMF).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 2.78 (4 H, m, O Su ), 3.01 and 3.25 (2 H, two dd, C ^β H Trp), 3.98 ( 1 H, m, C ^α H Trp), 4.97 (2 H, s, -OC H ₂ C ₆ H ₅ ), 6.73-7.62 (10 H, m, -OCH ₂ C ₆ H ₅ , indole), 8.56 ( 1 H, d, NH Trp), 10.78 (1 H, s, NH indol).

g) N-carbobenzoxy-L-tryptophanyl-L-leucine amide, ZL-Trp-L-Leu-NH ₂ .

3.30 g (13.52 mmol) of TFA * L-IleNH _{2 was} dissolved in 30 ml of DMF with the addition of 2.35 ml (13.52 mmol) of DIPEA. The reaction mixture was stirred with amine for half an hour. Then, with stirring, a solution of 7.06 g (16.22 mmol 20% excess) of ZL-TrpOSu in 30 ml of DMF was added. Stirred for 12 h at room temperature. At the end of the reaction (TLC control in system A, UV and tolidine sample), the solution was evaporated to the state of a mobile oil, dissolved in 200 ml of ethyl acetate, extracted with 3% H ₂ SO ₄ (1 × 100 ml), then with 5% NaHCO ₃ solution (2 × 100 ml) and distilled water (1 × 100 ml), the organic phase was dried over Na ₂ SO ₄ , the desiccant was filtered off, the filtrate was evaporated. The resulting oil was dissolved in a minimum amount of DMF, diluted with 200 ml of distilled water, and a white precipitate was obtained. The precipitate was allowed to stand for 12 h in a refrigerator at + 5 ° С, filtered off, washed with distilled water and hexane. Dried in a vacuum desiccator over CaCl ₂ and paraffin. The yield was 6.78 g (%) of pure product with mp. 166-169 ° C, [α] ^D ₂₆ = -31 ° (c = 1, DMF).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.08-1.22 (1 H, m , C ^γ H ₂ Leu), 1.45 (2 H, m, C ^β H Leu), 2.90 and 3.09 (2 H, two dd, C ^β H Trp), 4.28- 4.31 (2 H, m, C ^α H Leu and C ^α H Trp), 4.93 (2 H, m, CH ₂ CO), 6.99-7.30 (10 H, m, Ar) 7.25 and 7.33 (2 H, two s., NH ₂ amide), 7.62 (1 H, d, NH Leu), 7.97 (1 H, d, NH Trp), 10.81 ( 1 H, s, NH indol).

h) L-tryptophanyl-L-leucine amide, HL-Trp-L-Leu-NH ₂ .

In a flask containing a suspension of 0.500 g of 10% Pd / C in 50 ml of methanol was placed 6.78 g (0.015 mol) of ZL-Trp-L-Leu-NH ₂ . A stream of hydrogen was passed through the resulting suspension from a gasometer for 2 hours with vigorous stirring. At the end of the reaction, the catalyst was filtered through a fine-pored glass filter, placing a black ribbon marking paper on the bottom of the filter, washed with 50 ml of methanol, and the filtrate was evaporated. Received the product in the amount of 4.71 g (99%) in the form of a gray foam without a clear melting point, [α] ^D ₂₆ = -25 ° (c = 1, DMF).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm:, 0.82-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.09- 1.26 (1 H, m, C ^γ H ₂ Leu), 1.45 (2 N, m, C ^β H Leu), 2.90 and 3.08 (2 H, two dd, C ^β H Trp), 3.50 (1 H, m, C ^α H Trp), 4.14 (1 H, dd, C ^α H Leu), 6.94-7.55 (5 H, m, Ar), 7.05 and 7.41 (2 H, 2 d, NH ₂ amide), 7.60 (1 H, d, NH Leu), 8.05 (1 H, d, NH Trp), 10.85 (1 H, br. s, N indol).

i) Phenylpropionic acid succinimide ester, Ph (CH ₂ ) ₂ C (O) Su.

10.00 g Ph- (CH ₂ ) ₂ C (O) -OH (66.7 mmol) was dissolved in 150 ml of THF. To the solution was added 8.97 g (79.0 mmol, 18% excess) of N-hydroxysuccinimide, followed by 16.07 g (79.0 mmol, 18% excess) of dicyclohexylcarbodiimide. The reaction was carried out for 12 h with stirring at room temperature. The reaction progress was monitored by TLC in system A, (UV and toledine test). The dicyclohexylurea precipitate was filtered off. The solvent was evaporated, and a white precipitate began to form, it was triturated with hexane, decanted, and the remaining hexane was evaporated. An easily crystallizing oil was obtained which was dissolved in 200 ml of chloroform. Was extracted with a 5% aqueous solution of NaHCO ₃ (2 × 200 ml), then once with distilled water. Dried over anhydrous Na ₂ SO ₄ for half an hour. The desiccant was filtered off, washed with a small amount of chloroform, and chloroform was evaporated. The product was obtained in the form of white crystals, dried in air at room temperature. The yield was 7.42 g (45%) of the product with mp. 83-84 ° C.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 2.81 (4 H, m, OSu), 2.97-3.01 (2 H, m, CH ₂ C ₆ H ₅ ) , 3.34 (2 H, s, CH ₂ CO), 7.16-7.31 (5 H, m, Ar).

j) N-phenylpropionyl-L-tryptophanyl-L-leucine amide, Ph- (CH ₂ ) ₂ -C (O) -L-Trp-L-Leu-NH ₂ (I).

4.50 g (14.3 mmol) of HL-Trp-L-Leu-NH _{2 was} dissolved in 50 ml of DMF, 3.86 g (15.6 mmol, 10% ex) of succinimide phenylpropionic acid ester was added, the mixture was stirred for 12 h.Then 0.20 ml of DMAPA was added to remove excess Ph (CH ₂ ) ₂ C (O) OSu, stirred for another half hour, then the solvent was evaporated in vacuum at a temperature not higher than + 50 ° C. The resulting oil was diluted with 200 ml of distilled water, and a white “milky” solution was obtained. The solution was allowed to stand (“ripen”) for 24 h in a refrigerator at a temperature of + 5 ° C, a white powdery precipitate was obtained, which was then filtered off. The crystals obtained were washed with 100 ml of warm (43-45 ° C) water and 100 ml of hexane and 50 ml of diethyl ether. It was dried in an evacuated desiccator over CaCl ₂ and paraffin. The yield was 4.49 g (70%) of the product in the form of a white powder, mp. 197-198 ° C, [α] _D ²⁶ = -27 ° (c = 1, DMF), R _f 0.73 (CHCl ₃ : MeOH 9: 1).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.45 (2 H, t, C ^β H Leu), 1.56-1.73 (1 H, m, C ^γ H ₂ Leu), 2.38 (2 H, t, CH ₂ C ₆ H ₅ ), 2.69 (2 H, t, CH ₂ CO), 2.90 and 3.09 (2 H, two dd, C ^β H Trp), 4.22 (1 H, dd, C ^α H Leu), 4.54 (1 H, m, C ^α H Trp), 6.99-7.20 (10 H, m, Ar), 7.33 and 7.60 (2 H, two s, NH ₂ amide), 7.93 (1 H, d, NH Leu), 8.08 (1 H, d, NH Trp), 10.78 (1 H, s, NH indol).

EXAMPLE 2. Preparation of N-phenylpropioyl-D-tryptophannl-L-leucine (II) amide, C ₆ H ₅ (CH ₂ ) ₂ -C (O) -D-Trp-L-Leu-NH ₂

a) N-carbobenzoxy-L-tryptophan succinimide ester, Z-D-Trp-OSu.

To a solution of 10.00 g (29.6 mmol) of ZD-TrpOH (GL Biochem, Shanghai) in 300 ml of ethyl acetate was added 3.98 g (34.6 mmol, 17% excess) of N-hydroxysuccinimide, the solution was cooled to + 5 ° С, and then 7.25 ( 35.2 mmol, 19% excess) of dicylohexylcarbodiimide, taking care that the temperature does not exceed + 5 ° C. The reaction mixture was stirred for 30 min at a temperature of +5 - + 7 ° C and then for 12 h at room temperature. The dicyclohexylurea precipitate was filtered off, the filtrate was evaporated. The resulting viscous oil was dissolved in 200 ml of dichloromethane. The solution was kept for 24 h in a refrigerator at + 8 ° С, the re-formed precipitate of dicyclohexylurea was filtered off, and the filtrate was evaporated. The resulting oil was washed with hexane, the hexane was decanted, the oil was evaporated to dryness. The product was obtained in the amount of 11.84 g (92%) in the form of a creamy foam, [δ] ²⁵ _D + 44 ° (c 1, DMF).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 2.80 (4 H, m, O Su ), 3.07 and 3.25 (2 H, two dd, C ^β H Trp), 4.00 ( 1 H, m, C ^α H Trp), 4.95 (2 H, s, -OC H ₂ C ₆ H ₅ ), 6.95-7.77 (10 H, m, -OCH ₂ C ₆ H ₅ , indole), 8.56 ( 1 H, d, NH Trp), 10.80 (1 H, s, NH indol).

b) N-carbobenzoxy-D-tryptophanyl-L-leucine amide, ZD-Trp-L-Leu-NH ₂ .

3.30 g (13.52 mmol) of TFA * L-IleNH ₂ (prepared as in Id) was dissolved in 30 ml of DMF with the addition of 2.35 ml (13.52 mmol) of DIPEA. The reaction mixture was stirred with amine for half an hour. Then, with stirring, a solution of 7.06 g (16.22 mmol 20% excess) of ZD-TrpOSu in 30 ml of DMF was added. Stirred for 12 h at room temperature. At the end of the reaction (TLC control in system A, UV and tolidine sample), the solution was evaporated to the state of a mobile oil, dissolved in 200 ml of ethyl acetate, extracted with 3% H ₂ SO ₄ (1 × 100 ml), then with 5% NaHCO ₃ solution (2 × 100 ml) and distilled water (1 × 100 ml), the organic phase was dried over Na ₂ SO ₄ , the desiccant was filtered off, the filtrate was evaporated. The resulting oil was dissolved in a minimum amount of DMF, diluted with 200 ml of distilled water, and a white precipitate was obtained. The precipitate was allowed to stand for 12 h in a refrigerator at + 5 ° С, filtered off, washed with distilled water and hexane. Dried in a vacuum desiccator over CaCl ₂ and paraffin. The yield was 6.78 g (86%) of pure product with mp. 166-169 ° C, [α] ^D ₂₆ = -31 ° (c = 1, DMF).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.08-1.22 (1 H , m, C ^γ H ₂ Leu), 1.45 (2 H, m, C ^β H Leu), 2.90 and 3.09 (2 H, two dd, C ^β H Trp), 4, 28-4.31 (2 H, m., C ^α H Leu and C ^α H Trp), 4.93 (2 H, m, CH ₂ CO), 6.99-7.30 (10 N, m, Ar) 7.25 and 7.33 (2 H, two s, NH ₂ amide), 7.62 (1 H, d, NH Leu), 7.97 (1 H, d, NH Trp), 10.81 (1 H, s, NH indol).

c) D-tryptophanyl-L-leucine amide, HD-Trp-L-Leu-NH ₂ .

In a flask containing a suspension of 0.500 g of 10% Pd / C in 50 ml of methanol was placed 6.78 g (0.015 mol) of ZD-Trp-L-Leu-NH ₂ . A stream of hydrogen was passed through the resulting suspension from a gasometer for 2 hours with vigorous stirring. At the end of the reaction, the catalyst was filtered through a fine-pored glass filter, placing a black ribbon marking paper on the bottom of the filter, washed with 50 ml of methanol, and the filtrate was evaporated. Received the product in the amount of 4.71 g (99%) in the form of a gray foam with so pl. 108 ° C.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.09-1.26 (1 H , m, C ^γ H ₂ Leu), 1.45 (2 N, m, C ^β H Leu), 2.75 and 3.11 (2 H, two dd, C ^β H Trp), 3.50 (1 N, m , С ^α Н Trp), 4.14 (1 Н, dd, С ^α Н Leu), 6.94-7.55 (5 Н, m, indole), 7.05 and 7.41 (2 Н, 2 d, NH ₂ amide) 7.95 (1 H, d, NH Leu), 10.85 (2 H, br s, NH ₂ Trp)

d) N-phenylpropionyl-D-tryptophanyl-L-leucine amide, Ph (CH ₂ ) ₂ C (O) -D-Trp-L-Leu-NH ₂

4.50 g (14.3 mmol) of HD-Trp-L-Leu-NH _{2 was} dissolved in 50 ml of DMF, 3.86 g (15.6 mmol, 10% eq.) Of phenylpropionic acid succinimide ester was added, the mixture was stirred for 12 h. Then 0.20 ml of DMAPA was added to remove excess Ph (CH ₂ ) ₂ C (O) OSu, stirred for another half hour, then the solvent was evaporated in vacuum at a temperature not higher than + 50 ° C. The resulting oil was diluted with 200 ml of distilled water, and a white “milky” solution was obtained. The solution was allowed to stand (“ripen”) for 24 h in a refrigerator at a temperature of + 5 ° C, a white powdery precipitate was obtained, which was then filtered off. The crystals obtained were washed with 100 ml of warm (43-45 ° C) water and 100 ml of hexane and 50 ml of diethyl ether. It was dried in an evacuated desiccator over CaCl ₂ and paraffin. The yield was 4.49 g (70%) of the product in the form of a white powder, mp. 190 ° C, [α] _D ²⁶ = -22 ° (c = 1, DMF), R _f 0.73 (CHCl ₃ : MeOH 9: 1).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.45 (2 H, t, C ^β H Leu), 1.56-1.73 (1 H, m, C ^γ H ₂ Leu), 2.38 (2 H, t, CH ₂ C ₆ H ₅ ), 2.69 (2 H, t, CH ₂ CO), 2.90 and 3.09 (2 H, two dd, C ^β H Trp), 4.22 (1 H, dd, C ^α H Leu), 4.54 (1 H, m, C ^α H Trp), 6.99-7.20 (10 H, m, Ar), 7.33 and 7.60 (2 H, two s, NH ₂ amide), 7.93 (1 H, d, NH Leu), 8.08 (1 H, d, NH Trp), 10.78 (1 H, s, NH indol).

EXAMPLE 3. Preparation of N-phenylpropionyl-L-tryptophanyl-D-isoleucine (III) amide, C ₆ H ₅ (CH ₂ ) ₂ -C (O) -L-Trp-D-Leu-NH ₂

a) N-tert-butyloxycarbonyl-L-leucine, N-Boc-D-Leu-OH.

Dissolved 8.00 g (60.98 mmol) of D-leucine in a mixture of 50 ml of 1N NaOH, 100 ml of water, 200 ml of isopropyl alcohol. After complete dissolution, 15.97 g (73.17 mmol, 20% excess) of di-tert-butylpyrocarbonate were added. The mixture was stirred for 2 h at room temperature, maintaining pH = 9-10 by dropwise addition of a 1N NaOH solution. The reaction progress was monitored by TLC in system A, development with a chloro-tolidine sample. At the end of the reaction, the reaction mass was evaporated in a vacuum rotary evaporator to one third of the volume, while isopropyl alcohol was mainly removed. Excess di-tert-butylpyrocarbonate was extracted with hexane (2 × 100 ml). The aqueous solution was acidified with 10% H ₂ SO ₄ solution to pH 4, the product precipitated as a white "curd" precipitate. The precipitate was allowed to stand for 12 h, then filtered off, washed with distilled water until neutral, and dried in air in a porcelain cup to constant weight. Received 11.14 g (79%) of the product in the form of a white powder with so pl. 83 ° C.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.85 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.08-1.22 (1 H , m, C ^γ H ₂ Leu), 1.36 (9 H, s, -OC (CH ₃ ) ₃ ), 1.45 (2 H, m, C ^β H Leu), 3.86-3.88 (1 H, dd, C ^α H Leu), 6.72 (1 H, d, NH Leu), 6.89 and 7.19 (2 H, two s, NH ₂ amide).

b) N-tert-butyloxycarbonyl-D-leucine succinimide ester, N-Boc-D-Leu-OSu.

To a solution of 8.00 g (34.63 mmol) of Boc-D-leucine in 150 ml of THF was added 4.73 g (39.88 mmol, 15% excess) of N-hydroxysuccinimide, the solution was cooled to + 5 ° C in a water bath with ice, then 8.26 g (39.88 mmol, 15% excess) of dicylohexylcarbodiimide was added. The reaction mixture was stirred for 30 min at + 5 ° С and for 5 h at room temperature. The progress of the reaction was monitored by TLC in system A. At the end of the reaction, the precipitate of dicyclohexylurea was filtered off, the filtrate was evaporated. The resulting viscous oil was dissolved in 30 ml of dichloromethane. The solution was kept for 24 h in a refrigerator at + 8 ° С, the re-formed precipitate of dicyclohexylurea was filtered off, the organic phase was washed with 5% NaHCO ₃ (2 × 100 ml) and 100 ml of distilled water. It was dried over Na ₂ SO ₄ , the desiccant was filtered off, the filtrate was evaporated, and a thick white oil was obtained, which was crystallized in isopropyl alcohol. The crystals obtained were kept for 12 h in a refrigerator at + 8 ° C, the product was obtained in the amount of 8.31 g (73%) in the form of a glassy substance with so pl. 110-112 ° C, [α] _D ²⁶ = + 39 ° (c = 2, dioxane)

¹ H-NMR (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.39 (9 H, s, -OC (CH ₃ ) ₃ ), 1.45 (2 H, t, C ^β H Leu), 1.56-1.73 (1 N, m, C ^γ H ₂ Leu), 2.82 (4 N, m , OSu), 3.86-3.88 (1 H, m, C ^α H Leu), 7.93 (1 H, d, NH Leu).

c) N-tert-butyloxycarbonyl-D-leucine amide, N-Boc-D-Leu-NH ₂ .

To a solution of 8.00 g (24.36 mmol) Boc-D-Leu-OSu in 30 ml of DMF was added 100 ml of an aqueous ammonia solution with stirring; a white curdled precipitate immediately formed. The resulting suspension was stirred for 30 min until the reaction was complete. The precipitate was allowed to stand for 3 h in a cool place, but not in a refrigerator, then the formed precipitate was filtered off on a glass filter, washed with distilled water until the wash water was neutral, and dried in an oven at 100 ° C until constant weight. Received 3.37 g (60%) Boc-L-Leu-NH ₂ in the form of a white powder with so pl. 148-149 ° С, [α] _D ²⁵ (lit. data 150 ° С)

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.85 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.08-1.22 (1 H , m, C ^γ H ₂ Leu), 1.36 (9 H, s, -OC (CH ₃ ) ₃ ), 1.45 (2 H, m, C ^β H Leu), 3.86-3.88 (1 H, dd, C ^α H Leu), 6.72 (1 H, d, NH Leu), 6.89 and 7.19 (2 H, two s, NH ₂ amide).

d) D-leucine amide trifluoroacetate, TFA * D-Leu-NH ₂

To a suspension of 5.12 g (22.3 mmol) Boc-D-Leu-NH ₂ in 20 ml of dichloromethane was poured 26 ml (330 mmol, 150% excess) TFA, stirred for 2 hours. The solution was evaporated with diethyl ether (3 × 50 mL). The resulting white oil was triturated under diethyl ether to obtain a white powder, the ether was decanted, the residue was filtered. The product was air dried. Received 5.37 g (98%) TFA * D-Leu-NH ₂ in the form of slightly yellowish crystals spreading in air without a clear melting point, [α] _D ²⁶ = + 44 ° (c = 1, DMF).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.83-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.08-1.22 (1 H, m, C ^γ H ₂ Leu), 1.36 (9 H, s, -OC (CH ₃ ) ₃ ), 1.45 (2 H, m, C ^β H Leu), 3.69 (1 H, d d, C ^α H Leu), 7.52 and 7.93 (2 H, two s, NH ₂ amide), 8.12 (3 H, br.s, N ⁺ H ₃ Leu).

e) L-tryptophanyl-D-leucine amide, H-LTrp-D-Leu-NH ₂ .

In a flask containing a suspension of 0.500 g of 10% Pd / C in 50 ml of methanol was placed 6.78 g (0.015 mol) of ZL-Trp-D-Leu-NH ₂ . A stream of hydrogen was passed through the resulting suspension from a gasometer for 2 hours with vigorous stirring. At the end of the reaction, the catalyst was filtered through a fine-pored glass filter, placing a black ribbon marking paper on the bottom of the filter, washed with 50 ml of methanol, and the filtrate was evaporated. Received the product in the amount of 4.71 g (99%) in the form of a gray foam with so pl. 110 ° C.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (6 H, 2 d d, 2C ^δ H ₃ Leu), 1.09-1.26 (1 H , m, C ^γ H ₂ Leu), 1.45 (2 N, m, C ^β H Leu), 2.75 and 3.11 (2 H, two dd, C ^β H Trp), 3.50 (1 N, m , С ^α Н Trp), 4.14 (1 Н, dd, С ^α Н Leu), 6.94-7.55 (5 Н, m, indole), 7.05 and 7.41 (2 Н, 2 d, NH ₂ amide) 7.95 (1 H, d, NH Leu), 10.85 (2 H, br s, NH ₂ Trp)

f) N-phenylpropionyl-L-tryptophanyl-D-leucine amide, Ph (CH ₂ ) ₂ C (O) -L-Trp-D-Leu-NH ₂

4.50 g (0.0143 mol) of HL-Trp-D-Leu-NH _{2 was} dissolved in 50 ml of DMF, 3.86 g (0.0156 mol, 10% ex.) Of succinimide phenylpropionic acid ester Ph (CH ₂ ) ₂ C (O) were added OSu (obtained as in Ii) was stirred for 24 h. Then 0.20 ml of DMPDA was added to remove excess Ph (CH ₂ ) ₂ C (O) OSu, stirred for another half hour, then the solvent was evaporated in vacuum at a temperature not higher than +45 ° C. The resulting oil was diluted with 200 ml of distilled water, and a white "milky" precipitate was obtained. The precipitate was allowed to stand (form) for 12 h in a refrigerator at a temperature of + 8 ° C, then was filtered off. The crystals obtained were washed with 100 ml of warm (43 ° C) water and 100 ml of hexane and 50 ml of diethyl ether. Dried in an evacuated desiccator over CaCl ₂ . The yield was 4.49 g (70%) of the product in the form of a white powder, mp. 197-198 ° C, [α] _D ²⁶ = -27 ° (c = 1, DMF), R _f 0.73 (CHCl ₃ : MeOH 9: 1).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (6 H, m, 2C ^δ H ₃ Leu), 1.45 (2 H, t, C ^β H Leu), 1.56-1.73 (1 H, m, C ^γ H ₂ Leu), 2.38 (2 H, t, CH ₂ C ₆ H ₅ ), 2.69 (2 H, t, CH ₂ CO), 2.90 and 3.09 (2 H, two dd, C ^β H Trp), 4.22 (1 H, dd, C ^α H Leu), 4.54 (1 H, m, C ^α H Trp), 6.99-7.20 (10 H, m, Aryl), 7.33 and 7.60 (2 H, two s, NH ₂ amide), 7.93 (1 H, d, NH Leu ), 8.08 (1 H, d, NH Trp), 10.78 (1 H, s, NH indol).

EXAMPLE 4. Preparation of phenylpropionyl-L-tryptophanyl-L-leucine methyl ester, N-Ph (CH ₂ ) ₂ C (O) -L-Trp-L-Leu-OMe (IV)

a) L-leucine methyl ester hydrochloride, HCl * L-Leu-OMe

In a three-necked round-bottom flask equipped with a thermometer, dropping funnel, and an air cooler with a chlorine-kalydium tube, 150 ml of anhydrous methanol (dried over sieves with a pore size of 3 Å) was added, and the flask was cooled to -20 ° C in a bath with alcohol and liquid nitrogen. With stirring, 7.67 ml (106.70 mmol) of thionyl chloride was added dropwise. During the addition, the temperature of the reaction mixture was maintained at -20 ° to -15 ° C. After the addition of all thionyl chloride, 7.00 g (53.35 mmol) of L-leucine was added to the reaction flask (simultaneously, but taking care that the temperature of the reaction mixture did not rise above -15 ° C). The reaction was carried out at -15 ° C for 10 min, then allowed to warm to -5 ° C, after which the reaction was carried out at this temperature for another 2 h. The reaction mass was heated to + 40 ° C using a water bath with a controlled temperature, the reaction was continued for 8 h. After the completion of the reaction (TLC control in system B), the mixture was evaporated, and white glassy crystals were obtained, they were re-evaporated with methanol (2 × 150 ml), then triturated under 50 ml of anhydrous (kept over CaCl ₂ ) diethyl ether, ether was decanted, the residue was filtered off. The resulting product was dried in an evacuated desiccator over KOH. A white powder of L-leucine methyl ester hydrochloride weighing 8.96 g (92.4%) was obtained, mp. 147-149 ° C, [α] ^D ₂₆ = -13.8 ° (c = 5, H ₂ O), (lit. data: mp 150 ° C, [α] ^D ₂₅ = -13.4 °, c = 5, H ₂ O [AA Gershkovich, VK Kibirev. Chemical synthesis of peptides. Kiev: Naukova Dumka, 1992]).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.86 (6 H, m, 2C ^δ H ₃ Leu), 1.64-1.67 (3 H, m , C ^β H Leu, C ^γ H ₂ Leu), 3.72 (3 H, s, -OCH ₃ ), 3.88 (1 H, dd, C ^α H Leu), 8.75 (3 H, br.s, N ⁺ H ₃ Leu)

b) N-carbobenzoxy-L-tryptophanyl-L-leucine methyl ester, Z-L-Trp-L-Leu-OMe.

2.43 g (13.36 mmol) of HCl * L-Leu-OMe was dissolved in 30 ml of DMFA with 2.54 ml (14.57 mmol, 10% excess) of DIPEA, 6.98 g (16.03 mmol, 20% excess) of ZL-Trp-OSu was added dropwise to this solution obtained as in I (e) in 50 ml of DMF. The reaction was carried out for 24 hours at room temperature (TLC-control in system A, R _f 73). The reaction mass was evaporated to the state of a mobile oil, diluted with 300 ml of EA, this solution was washed with 3% H ₂ SO ₄ solution (2 × 300 ml) then 5% NaHCO ₃ solution (2 × 300 ml) and distilled water (1 × 300 ml) ... The organic fraction was separated, dried over anhydrous MgSO ₄ for half an hour, evaporated to the state of a mobile oil, 200 ml of distilled water at room temperature was added to it, a “milky” precipitate was obtained, water was evaporated to dryness, the product was dried in a vacuum desiccator over CaCl ₂ and paraffin. 5.95 g (95.6%) of the substance was obtained in the form of a thick oil interspersed with crystals without a clear melting point, [α] _D ²⁶ = -21.8 ° (c = 1, DMFA) (Ref. Data: mp 55-57 ° С [I. Bhardwaj, R. Sapra, V. Haridas. Urea-cored peptides for anion binding and vesicle formation. Arkivoc 2016 (6) 184-197], [α] _D ²⁴ = -28 ° (c = 0.1, acetonitrile) [V. Haridas; S. Sadanandan; S. Dhawan; R. Mishra; I. Jain; G. Goel; Hu Yuan; S. Patel. Synthetic minimalistic tryptophan zippers as a chiroptical switch. Org. Biomol. Chem .; vol. 15; no. 7; (2017); p. 1661-1669]).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.83-0.88 (6 H, m, 2C ^δ H ₃ Leu), 1.06-1.35 (2 H, t, C ^β H Leu), 1.58 (1 H, m, C ^γ H ₂ Leu), 2.90 and 3.06 (2 H, two d.d., C ^β H Trp), 3.63 (3 H, s, -OCH ₃ ), 4.29 (1 H, d d., C ^α H Leu), 4.41 (1 H, m, C ^α H Trp),), 4.96 and 5.02 (2 H, 2 d, -OC H ₂ C ₆ H ₅ ), 6.98-7.51 (10 H, m, -OCH ₂ C ₆ H ₅ , indole), 7.67 (1 H, d, NH Trp), 8.27 (1 H, d, NH Leu), 10.83 (1 H, s, NH indol)

c) L-tryptophanyl-L-leucine methyl ester, H-L-Trp-L-Leu-OMe.

In a flask containing a suspension of 1.00 g of 10% Pd / C with 100 ml of methanol and 1 ml of distilled water, 5.79 g (12.44 mmol) of ZL-Trp-L-Leu-OMe were placed. Hydrogen was passed through the resulting suspension for 2 hours with vigorous stirring. The progress of the reaction was monitored by TLC in system A ( _{product R f} 0.35). At the end of the reaction, the catalyst was filtered through a glass filter, lined with black ribbon paper, and washed with methanol. The filtrate was evaporated to give 4.15 g of the product as a white thick oil with [α] _D ²⁶ = -28.6 ° (c = 1, DMFA).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.83-0.88 (6 H, m, 2C ^δ H ₃ Leu), 1.06-1.35 (2 H, t, C ^β H Leu), 1.58 (1 H, m, C ^γ H ₂ Leu), 2.90 and 3.06 (2 H, two d.d., C ^β H Trp), 3.63 (3 H, s, -OCH ₃ ), 4.29 (1 H, d d., C ^α H Leu), 4.41 (1 H, m, C ^α H Trp), 6.98-7.51 (10 H, m, -OCH ₂ C ₆ H ₅ , indole), 7.67 (1 H, d, NH Trp), 8.27 (1 H, d, NH Leu), 10.83 (1 H, s, NH indol)

d) N-phenylpropionyl-L-tryptophanyl-L-leucine methyl ester, N-Ph (CH ₂ ) ₂ C (O) -L-Trp-L-Leu-OMe (IV)

To 4.10 g (12.37 mmol) of HL-Trp-L-Leu-OMe in 70 ml of DMFA was added 3.67 g (14.85 mmol, 20% excess) of phenylpropionic acid succinimide ester. The solution was stirred for 12 h. The progress of the reaction was monitored by TLC in system A. An excess amount of succinimide ether was quenched by adding 0.2 ml of DMAPA. The solvent was evaporated, diluted with 300 ml of EA. It was extracted with a 3% solution of H ₂ SO ₄ (1 × 300 ml), then with a 5% solution of NaHCO ₃ (2 × 300 ml), then with a saturated solution of NaCl (1 × 300 ml) and distilled water (1 × 300 ml). The organic fraction was dried over anhydrous MgSO ₄ , evaporated to dryness, an easily crystallizing oil was obtained, it was evaporated twice with diethyl ether until a stable foam was obtained. The foam was crushed, transferred to a glass filter and washed with hexane. The product was dried in a vacuum desiccator over Na ₂ SO ₄ and paraffin. Received 4.25 g (74%) of the substance in the form of a white powder with so pl. 134-135 ° C, [α] _D ²⁶ = -41.4 °, s = 1, DMFA

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.83-0.88 (6 H, m, 2C ^δ H ₃ Leu), 1.06-1.35 (2 H, t, C ^β H Leu), 1.58 (1 H, m, C ^γ H ₂ Leu), 2.34 (2 H, m, CH ₂ chains), 2.69 (H, m, CH ₂ chains), 2.92 and 3.08 (2 H, two dd, C ^β H Trp), 3.61 (3 H, s, -OCH ₃ ), 4.32 (1 H, dd, C ^α H Leu), 4.61 (1 H, m, C ^α H Trp), 6.91- 7.61 (10 H, m, aryl), 8.02 (1 H, d, NH Trp), 8.35 (1 H, d, NH Leu), 10.91 (1 H, s, NH indol)

EXAMPLE 5 Obtaining phenylpropionyl-L-tryptophanyl-L-leucine, Ph (CH ₂ ) ₂ C (O) -L-Trp-L-Leu-OH (V).

a) N-phenylpropionyl-L-tryptophanyl-L-isoleucine, N-Ph (CH ₂ ) ₂ C (O) -L-Trp-Leu-OH.

1.10 g (2.32 mmol) of N-Ph (CH ₂ ) ₂ C (O) -L-Trp-L-Leu-OMe, obtained as in IV (d), was dissolved in 10 ml of methanol, solution 0 was added with stirring, 19 g (4.64 mmol, 100% excess) NaOH in 5 ml of water. Stirred for 3 hours until complete dissolution of the reaction mass. The reaction mass was diluted with 100 ml of water, acidified with 10% H ₂ SO ₄ to pH 4-5 with stirring, and a “milky” precipitate formed, which was allowed to stand for 12 h in a refrigerator at + 5 ° C. The solution over the formed solid product was decanted, the product was dissolved in 100 ml of ethyl acetate. The organic phase was extracted with distilled water (2 × 100 ml), dried over anhydrous Na ₂ SO ₄ for half an hour. The desiccant was filtered off, washed with a small amount of ethyl acetate, evaporated, then re-evaporated 3 times with diethyl ether until a white stable foam was obtained. The foam was crushed, transferred to a glass filter and washed with hexane. The yield was 0.95 g (%) with mp. 91-92 ° C, [α] _D ²⁹ = -19.8 °, c = 1, DMFA.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.83-0.88 (6 H, m, 2C ^δ H ₃ Leu), 1.06-1.35 (2 H, t, C ^β H Leu), 1.58 (1 H, m, C ^γ H ₂ Leu), 2.34 (2 H, m, CH ₂ chains), 2.69 (H, m, CH ₂ chains), 2.92 and 3.08 (2 H, two dd, C ^β H Trp), 3.61 (3 H, s, -OCH ₃ ), 4.32 (1 H, dd, C ^α H Leu), 4.61 (1 H, m, C ^α H Trp), 6.91- 7.61 (10 H, m, aryl), 8.02 (1 H, d, NH Trp), 8.35 (1 H, d, NH Leu), 10.91 (1 H, s, NH indol) 12.63 (1 H, br s , HE).

EXAMPLE 6. Preparation of phenylpropionyl-L-tryptophanyl-L-leucine methylamide, Ph (CH ₂ ) ₂ C (O) -L-Trp-L-Leu-NHCH ₃ (VI).

Dissolved 1.10 g (2.37 mmol) N-Ph (CH ₂ ) ₂ C (O) -L-Trp-L-Leu-OMe, obtained as in IV (d) in 23 g of a solution of methylamine in ethyl alcohol, containing 5.7 g of pure methylamine. Left in a sealed container for 6 days at room temperature. The solution was evaporated, then re-evaporated twice with diethyl ether. Received 1.05 g (96%) of the substance in the form of a white powder with so pl. 201 ° C with decomposition, [α] _D ²⁹ = -16.6 °, c = 1, DMFA.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.83-0.88 (6 H, m, 2C ^δ H ₃ Leu), 1.06-1.35 (2 H, t, C ^β H Leu), 1.58 (1 H, m, C ^γ H ₂ Leu), 2.37 (2H, t, CH ₂ C (O)), 2.57 (3 H, d, -NH CH ₃ ), 2.69 (2H, t, CH ₂ C ₆ H ₅ ), 2.91 and 3.11 (2 H, two dd, C ^β H Trp), 4.12 (1 H, dd, C ^α H Leu), 4.35 (1 H, m, C ^α H Trp), 4.93 and 4.99 (2 H, 2 d, -OC H ₂ C ₆ H ₅ ), 6.91-7.28 (10 H, m, -OCH ₂ C ₆ H ₅ , indole), 7.60 (1 H, d , NH Trp), 7.85 (1 H, d, NH Leu), 10.83 (1 H, s, NH indol).

EXAMPLE 7. Preparation of N-phenylacetyl-L-tryptophanyl-L-leucine amide, PhCH ₂ C (O) -L-Trp-L-Leu-NH ₂ (VII).

a) Phenylacetic acid succinimide ester.

To a solution of 5.00 g (37.0 mmol) of phenylacetic acid in 50 ml of tetrahydrofuran was added 5.07 g (44.0 mmol, 20% excess) of N-hydroxysuccinimide, the solution was cooled to 0 ° C, and then within 5 min 9.08 g (44.0 mmol, 20% excess) of dicylohexylcarbodiimide was added, making sure that the temperature did not exceed + 5 ° C. The reaction mixture was stirred for 30 min at a temperature of 0 - + 5 ° С and for another 5 h at room temperature. At the end of the reaction (TLC control in system A, Rf 0.83), the dicyclohexylurea precipitate was filtered off, the filtrate was evaporated on a rotary evaporator. The resulting viscous oil was dissolved in 50 ml of dichloromethane. The solution was kept for 24 h in a refrigerator at + 8 ° C, the re-formed precipitate of dicyclohexylurea was filtered off, the filtrate was evaporated, the oil was triturated with hexane, the hexane was decanted, the residue was evaporated to dryness. Received pure product in the amount of 9.44 g (97%) in the form of a white powder with so pl. = 111-112 ° C.

1H-NMR spectrum (DMSO-d6) d, ppm: 2.81 (4 H, m, OSu), 4.10 (2 H, s, CH ₂ C ₆ H ₅ ), 7.20-7.42 (5 H, m, C ₆ H ₅ ).

b) N-phenylacetyl-L-tryptophanyl-L-leucine amide, PhCH ₂ C (O) -LTrp-L-Leu-NH ₂ (VII).

To 0.55 g (1.76 mmol) of L-tryptophanyl-L-leucine amide obtained as in I (h) in 20 ml of dimethylformamide was added 0.46 g (20% excess) of succinimide ester of phenylacetic acid. The solution was stirred for 24 hours. The reaction progress was monitored by TLC (system A, _Rf 0.55). The excess amount of succinimide ester was quenched by the addition of 0.065 ml DMAPA. Then the reaction mass was evaporated on a rotary evaporator to a mobile oil, which was dissolved in 100 ml of ethyl acetate. Extracted with 3% aqueous solution of H ₂ SO ₄ (2 × 100 ml), 5% aqueous solution of NaHCO ₃ (2 × 200 ml), and distilled water (once). The organic phase was separated, dried over anhydrous Na ₂ SO ₄ for half an hour. The drying agent was filtered off, washed with a small amount of ethyl acetate, and evaporated. It was dried in an evacuated desiccator over CaCl ₂ , the yield was 0.700 g (%) of the substance in the form of a white powder, mp. 123 ° C, [α] _D ²⁷ = -16 °, c = 0.5, DMFA.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm:, 0.82-0.88 (6 H, m, 2C ^δ H ₃ Leu), 1.45 (2 H, t, C ^β H Leu ), 1.56-1.73 (1 N, m, C ^γ H ₂ Leu), 2.95 and 3.09 (2 H, two dd, C ^β H Trp), 3.39 (2 H, s, CH ₂ С ₆ Н ₅ ), 4.15 (1 Н, dd, C ^α H Leu), 4.62 (1 Н, m, С ^α Н Trp), 6.97-7.38 (10 Н, m, СН ₂ С ₆ H ₅ , indole), 7.07 and 7.58 (2 H, 2 d, NH ₂ amide), 7.77 (1 H, d, NH Leu), 8.28 (1 H, d, NH Trp), 10.81 (1 H, s , NH indol).

EXAMPLE 8 Preparation of N-caproyl-L-tryptophanyl-L-leucine amide, CH ₃ (CH ₂ ) ₄ C (O) -L-Trp-L-Leu-NH ₂ (IIX).

a) Caproic (hexanoic) acid succinimide ester, CH ₃ (CH ₂ ) ₄ C (O) Su.

To a solution of 7.00 g (60.26 mmol) of caproic acid in 100 ml of THF was added 7.97 g (69.30 mmol, 15% excess) of N-hydroxysuccinimide, the solution was cooled to + 5 ° С, after which 13.66 g (66.29 mmol, 10% excess) dicylohexylcarbodiimide, making sure that the temperature does not exceed + 5- + 10 ° C. Then the reaction mass was stirred for 30 min at a temperature of + 5- + 10 ° C and for 5 h at room temperature. The dicyclohexylurea precipitate was filtered off, washed with a small amount of THF, and the solvent was evaporated on a rotary evaporator. An easily crystallizing oil was obtained, which was triturated with 50 ml of hexane, then the hexane was decanted. The resulting oil was triturated with diethyl ether, but the product did not crystallize. The ether was decanted, the oil was dissolved in 100 ml of chloroform, washed with 5% NaHCO ₃ solution (2 × 100 ml) and once with 100 ml of distilled water. The organic fraction was dried over calcined anhydrous Na ₂ SO ₄ for an hour, the desiccant was filtered off, and the solvent was evaporated. The pure product was obtained in the amount of 6.27 g (49%) as a light yellow wax without a clear melting point.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.87 (3 H, m, СН _{3 chains} ), 1.32 (2 Н, m, С ^δ Н ₂ ), 1.62 (4 Н, m, C ^γ H ₂ C ^β H ₂ ), 2.63-2.68 (2 H, t, C ^α H ₂ ), 2.80, (4 H, m, OSu).

b) N-caproyl-L-tryptophanyl-L-leucine amide, CH ₃ (CH ₂ ) ₄ C (O) -LTrp-L-Leu-NH ₂ (IIX).

To 0.55 g (1.73 mmol) of L-tryptophanyl-L-leucine amide obtained as in I (h) in 20 mL of DMFA was added 0.45 g (2.10 mmol, 20% excess) of caproic acid succinimide ester. The solution was stirred for 7 h. The progress of the reaction was monitored by TLC in system A, R _f 0.62. The excess amount of succinimide ester was quenched by the addition of 0.02 ml of DMAPA and stirred for another half hour. Then the reaction mass was diluted 3 times with distilled water and evaporated on a rotary evaporator. An easily crystallizing oil was obtained which was dissolved in 70 ml of ethyl acetate. This solution was extracted with 3% aqueous solution of H ₂ SO ₄ (2 × 70 ml), 5% aqueous solution of NaHCO ₃ (2 × 70 ml), then once with distilled water. The organic phase was separated, dried over calcined anhydrous Na ₂ SO ₄ for half an hour. The drying agent was filtered off, washed with a small amount of ethyl acetate, and evaporated. It was dried in an evacuated desiccator over CaCl ₂ ; the yield was 0.745 g (%) of the substance in the form of a white powder, mp. 166 ° C, [α] _D ²⁷ = -20 °, c = 1, DMFA.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.82-0.88 (9 Н, m, 2С ^δ H ₃ Leu and СН _{3 caproyl (chain)} ),), 1.32 (2 Н, m, C ^δ H ₂ ), 1.45 (2 H, t, C ^β H Leu), 1.56-1.73 (1 N, m, C ^γ H ₂ Leu), 1.62 (4 N, m, C ^γ H ₂ C ^β H ₂ ), 2.63-2.68 (2 H, t, C ^α H ₂ ), 2.95 and 3.09 (2 H, two d.d., C ^β H Trp), 3.39 (2 H, s , C H ₂ C ₆ H ₅ ), 4.15 (1 H, dd, C ^α H Leu), 4.62 (1 H, m, C ^α H Trp), 6.97-7.38 (10 N, m, CH ₂ C ₆ H ₅ , indole), 7.07 and 7.58 (2 H, 2 d, NH ₂ amide), 7.77 (1 H, d, NH Leu), 8.28 (1 H, d, NH Trp), 10.81 (1 H, s, NH indol).

EXAMPLE 9. Preparation of N-phenylpropionyl-glycyl-L-leucine amide, Ph (CH ₂ ) ₂ C (O) -Gly-L-Leu-NH ₂ (IX).

a) N-phenylpropionyl-glycine succinimide ester, Ph (CH ₂ ) ₂ C (O) -Gly-OSu.

1.33 g (11.58 mmol, 20% excess) of N-hydroxysuccinimide, then 2.38 g (11.58 mmol, 20% excess) of dicylohexylcarbodiimide were added. The reaction was carried out for 12 h at room temperature. The dicyclohexylurea precipitate was filtered off, washed with a small amount of THF, and the solvent was evaporated on a rotary evaporator. A readily crystallizing oil was obtained which was dissolved in 200 ml of ethyl acetate. Washed with 5% aqueous NaHCO ₃ solution (2 × 100 ml), then once with distilled water. The organic phase was dried over anhydrous Na ₂ SO ₄ for half an hour. The drying agent was filtered off, washed with a small amount of ethyl acetate, and evaporated. The product was obtained in the amount of 2.55 g (83%) in the form of white glassy crystals without a clear melting point.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 2.37 (2 H, t, CH ₂ CO), 2.69 (2 H, t, CH ₂ C ₆ H ₅ ), 3.56 (2 H, m, CH ₂ Gly), 2.80, (4 H, m, OSu).

b) N-phenylpropionyl-glycyl-L-leucine amide, Ph (CH ₂ ) ₂ C (O) -Gly-L-Leu-NH ₂ (IX).

Dissolved 2.51 g (10.29 mmol) of TFA * L-IleNH ₂ obtained as in 1 (g) in 30 ml DMFA with the addition of 2.00 ml (11.31 mmol, 10% excess) DIPEA. The reaction mixture was stirred with amine for half an hour. Then, with stirring, a solution of 3.39 g (11.31 mmol, 10% excess) Ph (CH ₂ ) ₂ C (O) -Gly-OSu in 30 ml of DMF was added. Stirred for 12 h at room temperature. At the end of the reaction (TLC control in system A, UV, and tolidine sample), the solution was evaporated to the state of a mobile oil, diluted with 200 ml of distilled water, an oily, easily crystallized precipitate was obtained; additional purification by extraction was not required. The precipitate was allowed to stand for 24 h in a refrigerator at + 5 ° C, filtered off, washed with distilled water and hexane. Dried in an evacuated desiccator over Na ₂ SO ₄ and paraffin. The yield was 1.50 g (46%) of the product in the form of a slightly yellowish powder, mp. 162 ° C, [α] ^D ₂₉ = -5.2 ° C = 1, DMFA.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.85 (6 H, m, 2C ^δ H ₃ Leu), 1.45 (2 H, t, C ^β H Leu), 1 , 56-1.73 (1 H, m, C ^γ H ₂ Leu), 2.37 (2 H, t, CH ₂ CO). 2.69 (2 H, t, CH ₂ C ₆ H ₅ ), 3.56 (2 H, m, CH ₂ Gly), 4.22 (1 H, dd, C ^α H Leu), 6.99-7, 20 (10 H, m, aryl), 7.33 and 7.60 (2 H, two s, NH ₂ amide), 7.93 (1 H, d, NH Leu).

EXAMPLE 10 N-phenylpropionyl-L-tryptophanyl-glycine amide, Ph (CH ₂ ) ₂ C (O) -L-Trp-Gly-NH ₂ (X).

a) N-tert-butyloxycarbonyl-glycine succinimide ester, N-Boc-Gly-OSu.

To a solution of 3.00 g (17.1 mmol) of N-Boc-glycine in 100 ml of EA, 2.31 g (20.0 mmol, 17% excess) of N-hydroxysuccinimide was added, the solution was cooled to + 5 ° С in a water bath with ice, then 3.70 g ( 18.5 mmol, 10% excess) of dicylohexylcarbodiimide. The reaction mixture was stirred for 30 min at + 5 ° С and for 5 h at room temperature. The progress of the reaction was monitored by TLC in system A. After the completion of the reaction, the precipitate of dicyclohexylurea was filtered off, the filtrate was evaporated. The resulting viscous oil was dissolved in 30 ml of dichloromethane. The solution was kept for 24 h in a refrigerator at + 8 ° С, the re-formed precipitate of dicyclohexylurea was filtered off, the organic phase was washed with 5% NaHCO ₃ (2 × 100 ml) and 100 ml of distilled water. It was dried over anhydrous MgSO ₄ , the desiccant was filtered off, the filtrate was evaporated, and evaporation gave the product in the form of lamellar crystals in an amount of 4.56 g (98%), mp. 158 ° C. (Ref: 168-170 ° C [Anderson, GW; Zimmerman, JE; Callahan, FM The use of esters of N-hydroxysuccinimide in peptide synthesis. J. Am. Chem. Soc, 1964, 86, 1839-1842]) ...

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 1.39 (9 H, s, -OC (CH ₃ ) ₃ ), 2.81 (4 H, m, OSu), 4.01-4.10 (2 H , dd, CH ₂ Gly), 7.46-7.50 (1 H, t, NH Gly).

b) N-tert-butyloxycarbonyl-glycine amide Boc-Gly-NH ₂

To a solution of 4 g (14.0 mmol) Boc-Gly-OSu in 20 ml of DMF was added 40 ml of an aqueous solution of ammonia. The resulting solution was stirred for 30 min, then evaporated to dryness. The resulting product was triturated under diethyl ether to give a thick, caramel-like yellow oil. The ether was decanted, the product was used further without additional purification.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 1.39 (9 H, s, -OC (CH ₃ ) ₃ ), 3.54 - 3.59 (2 H, dd, CH ₂ Gly), 7.10 (2 H, two s, NH ₂ ), 7.48-7.50 (1 H, t, NH Gly).

c) Glycine amide trifluoroacetate, TFA * Gly-NH ₂

To 3.65 g (21.0 mmol) Boc-Gly-NH₂ 10 ml of dichloromethane and 25.14 g (221 mmol, 1000% excess) of trifluoroacetic acid were added, stirred for 2 h. Then the solvents were evaporated, the resulting oil was re-evaporated three times with diethyl ether to remove excess trifluoroacetic acid. The product was obtained as a yellow oil, the yield was 4.13 g (98%). The product was used further without further purification.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 3.50 (2 H, m, CH ₂ Gly), 8.05 (3 H, br.s, N ⁺ H ₃ Gly), 7.10 and 7.21 ( 2 H, two s, NH ₂ ),

d) N-carbobenzoxy-L-tryptophanyl-glycine amide, ZL-Trp-Gly-NH ₂ .

Dissolved 1.25 g (9.0 mmol), TFA * Gly-NH ₂ in 30 ml of DMF with the addition of 3.51 ml of DIPEA, stirred for 30 min. To the resulting solution was added 4.0 g (1.08 mol, 20% excess) of ZL-Trp-OSu (prepared as in Ie), stirred at room temperature for 24 h. After the completion of the reaction (TLC control in system A, Rf = 0 , 51), 0.2 ml of DMAPA was added to neutralize the excess of succinimide ether, and the mixture was stirred for 30 min. The solvents were evaporated, the resulting oil was dissolved in 200 ml of ethyl acetate, extracted with 3% solution of H ₂ SO ₄ (1 × 200 ml), 5% solution of NaHCO ₃ (1 × 200 ml), distilled water (2 × 200 ml), the organic phase was separated , dried over anhydrous Na ₂ SO ₄ , the desiccant was filtered off, the filtrate was evaporated. To the still warm and easily mobile oil, 100 ml of distilled water was added, a white precipitate formed, which was filtered off and washed with water. The yield was 2.26 g (92%) of the product with so pl. 123 ° C, [α] _D ²⁷ = -37 ° (c = 0.5, DMF). (Ref. Data: mp 118-119 ° C [α] _D ²⁴ = + 4 ° (c = 0.5, acetic acid) [M. Bodanszky; JC Tolle; J. Seto; WH Sawyer; Synthesis and some pharmacological properties of [8-L-tryptophan] oxytocin J. Med. Chem. V. 23; No. 11; (1980); p. 1259-1261, or [α] _D ²³ = -26.4 ° (c = 1.8, DMF) [ Celik, I .; Abdel-Fattah; Ashraf, AA Convenient synthesis of C-terminal di- and tri-peptide amides from N-protected dipeptidoylbenzotriazoles. Tetrahedron, 2009, 65, 4923-4929])

¹ H-NMR spectrum (deuteroacetone) δ, ppm: 2.92 and 3.11 (2 H, two dd, C ^β H Trp), 3.40 and 3.32 (2H, 2 d d CH ₂ Gly), 4.34 ( 1 N, m, C ^α H Trp), 4.98 and 5.03 (2 H, 2 d, -OC H ₂ C ₆ H ₅ ), 6.52-7.77 (10 N, m, -OC H ₂ C ₆ H ₅ , indole ), 7.41 (1 H, d, NH Trp), 7.41 and 7.10 (2 H, two s, NH ₂ amide), 7.66 (1 H, d, NH Gly), 10.12 (1 H, s, NH indol) ...

e) L-tryptophanyl-glycine amide, HL-Trp-Gly-NH ₂

In a flask containing a suspension of 0.400 g of 10% Pd / C in 100 ml of methanol was placed 2.97 g (7.5 mmol) of ZL-Trp-Gly-NH ₂ . A stream of hydrogen was passed through the resulting suspension from a gasometer for 2 hours with vigorous stirring. At the end of the reaction, the catalyst was filtered through a fine-pored glass filter, placing a black ribbon marking paper on the bottom of the filter, washed with 50 ml of methanol, and the filtrate was evaporated. The product was obtained in the amount of 2.04 g (99%) as a gray foam without a clear melting point, which was used further without further purification.

f) N-phenylpropionyl-L-tryptophanyl-glycine amide, Ph (CH ₂ ) ₂ C (O) -L-Trp-Gly-NH ₂ (X).

4.50 g (14.3 mmol) of HL-Trp-Gly-NH _{2 was} dissolved in 50 ml of DMF, 3.86 g (15.6 mmol, 10% ex.) Of phenylpropionic acid succinimide ester (Ph (CH ₂ ) ₂ C (O) Su) obtained as in I (i), stirred for 24 h. Then, 0.20 ml of DMPDA was added to remove excess Ph (CH ₂ ) ₂ C (O) OSu, stirred for another half hour, then the solvent was evaporated. The resulting oil was diluted with 200 ml of distilled water, and a white "milky" precipitate was obtained. The precipitate was allowed to stand (form) for 24 hours in a refrigerator at a temperature of + 8 ° C, then was filtered off. The crystals obtained were washed with 100 ml of distilled water and 50 ml of hexane. Dried in an evacuated desiccator over CaCl ₂ . The yield was 4.49 g (70%) of the product in the form of a white powder, mp. 118 ° C, [α] _D ²⁶ = -33 ° (c = 1, DMF), R _f 0.73 (CHCl ₃ : MeOH 9: 1).

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 2.92 and 3.11 (2 H, two dd, C ^β H Trp), 2.37 (2 H, t, CH ₂ CO) , 2.69 (2 H, t, CH ₂ C ₆ H ₅ ), 3.40 (2 H, m, CH ₂ Gly), 4.34 (1 H, m, C ^α H Trp), 6.52-7.77 (10 H, m, Aryl, indole), 7.41 (1 H, d, NH Trp), 7.41 and 7.10 (2 H, two s, NH ₂ amide), 7.66 (1 H, d, NH Gly), 10.12 (1 H, s , NH indol).

The results of a pharmacological study of the claimed compounds

1. Influence of the claimed compounds on the behavior of BALB / c mice under emotional stress in the open field test.

The open field test is a common model for assessing the behavior of rodents under conditions of emotional stress, which is formed as a reaction to a new environment and a threatening situation. The technique of an illuminated "open field" is used in this work, in which the transfer of an animal from darkness to a brightly illuminated arena creates an additional stress factor based on the natural tendency of rodents to avoid brightly lit places (S.B. Seredenin, A.A. Vedernikov. Effect of psychotropic drugs on the behavior of inbred mice in an open field // Byull. Exp. Biol. Med. - 1979. - T. 88, No. 7, pp. 38-40).

The study used mice-male inbred lines BALB / c weighing 19-25 g (NPP "Nursery of laboratory animals FIBC RAS). The animals were kept in a laboratory vivarium under controlled environmental conditions (20-22 ° C and 30-70% relative humidity, 12-hour lighting cycle, 10-fold change in room air volume per hour), in plastic cages with a top cover made of stainless steel with a dust-free bed of wood shavings, 20 mice in each cage, with constant access to extruded briquetted feed GOST R 50258-92 [1993] and drinking water. Animals were divided into groups randomly, according to the criterion of body weight, with a deviation from the mean by no more than ± 10%. Before the experiment, the animals were kept in the experimental room in the "home" cages for 24 hours.

The compounds were prepared in the form of a suspension with Tween-80 and distilled water and were administered by a single intraperitoneal injection (i.p.). 30 minutes after intraperitoneal injection, the animal was first placed for 1 min in a dark chamber, and then - on one of the peripheral squares of the "open field", which is a white round arena 1 meter in diameter with white sides 50 cm high. The arena is uniformly illuminated 4 - by shadowless lamps of 75 W each, located at a height of 1 m above the surface of the field. The entire space of the arena is evenly divided by 4 concentric circles, which in turn are divided by radii into sectors so that the peripheral circle consists of 16 identical curvilinear squares. The animals were observed for 3 minutes, the number of crossed squares at the periphery (PA), in the central regions (CA), the number of entries to the center (C), and the number of vertical posts (VA) were recorded separately. The total number of squares crossed, together with the number of uprights, was designated as total activity (ODA).

The presence of anxiolytic action was judged by the identification of an activating effect on locomotor activity in animals with a freezing reaction in the open field test (BALB / c line).

The results were statistically processed using one-way analysis of variance (ANOVA, Kruskal-Wallis test) and nonparametric analysis for independent variables (Mann-Whitney U-test).

Under the conditions of emotional stress in the "open field" test with intraperitoneal (i / b) administration, the compound GD-102 at doses of 0.01-0.10 mg / kg showed a statistically significant increase in the peripheral, central and general motor activity of mice. BALB / c compared with control (Fig. 1, Table 2) At doses of 0.5, 1.0 and 5.0 mg / kg, no significant effect was observed.

Notes:

Data are presented as M (SD), where M is the arithmetic mean; SD is the standard deviation;

the number of animals in the group -8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

Under the conditions of emotional stress in the "open field" test with intraperitoneal injection, compound GD-118 at doses of 0.1-1.0 mg / kg showed a statistically significant increase in the peripheral, central and general motor activity of BALB / c mice compared to with control (table 3)

Notes:

Data are presented as M (SD), where M is arithmetic mean; SD is the standard deviation;

the number of animals in the group - 8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

Under conditions of emotional stress in the "open field" test with intraperitoneal injection of compound GD-123 at doses of 0.1-1.0 mg / kg, a statistically significant increase in the peripheral, central and general motor activity of BALB / c mice compared with control not found (table. 4)

Notes:

Data are presented as M (SD), where M is the arithmetic mean; SD is the standard deviation;

the number of animals in the group - 8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

Under conditions of emotional stress in the "open field" test with intraperitoneal administration, the compound GD-125 at doses of 0.5-1.0 mg / kg showed a statistically significant increase in the peripheral, central and general motor activity of BALB / c mice compared to with control (table 5)

Notes:

Data are presented as M (SD), where M is the arithmetic mean; SD is the standard deviation;

the number of animals in the group - 8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

Under the conditions of emotional stress in the "open field" test with intraperitoneal injection of compound GD-128 at a dose of 0.5 mg / kg, a statistically significant increase in peripheral, central and general motor activity of BALB / c mice was found in comparison with the control (Table . 6)

Notes:

Data are presented as M (SD), where M is the arithmetic mean; SD is the standard deviation;

the number of animals in the group - 8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

Under conditions of emotional stress in the "open field" test with intraperitoneal injection, compound GD-129 at a dose of 0.5 mg / kg showed a statistically significant increase in the peripheral, central, and general motor activity of BALB / c mice compared to controls (Table . 7)

Notes:

Data are presented as M (SD), where M is the arithmetic mean; SD is the standard deviation;

the number of animals in the group - 8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

Under the conditions of emotional stress in the "open field" test with intraperitoneal injection of compound GD-107 at doses of 0.1-1.0 mg / kg, a statistically significant increase in the peripheral, central and general motor activity of BALB / c mice was found. with control (table 8)

Notes:

Data are presented as M (SD), where M is the arithmetic mean; SD is the standard deviation;

the number of animals in the group - 8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

Under conditions of emotional stress in the "open field" test with intraperitoneal injection, compound GD-108 at doses of 0.5-1.0 mg / kg showed a statistically significant increase in the peripheral, central and general motor activity of BALB / c mice compared to with control (table 9)

Notes:

Data are presented as M (SD), where M is the arithmetic mean; SD is the standard deviation;

the number of animals in the group - 8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

Under conditions of emotional stress in the "open field" test with intraperitoneal injection, compound GD-116 at doses of 0.1-1.0 mg / kg showed a statistically significant increase in the peripheral, central, and general motor activity of BALB / c mice compared to with control (tab. 10)

Notes:

Data are presented as M (SD), where M is the arithmetic mean; SD is the standard deviation;

the number of animals in the group - 8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

Under the conditions of emotional stress in the "open field" test with intraperitoneal injection of compound GD-119 at doses of 0.1-1.0 mg / kg, a statistically significant increase in the peripheral, central and general motor activity of BALB / c mice was found. with control (table 11)

Notes:

Data are presented as M (SD), where M is the arithmetic mean; SD is the standard deviation;

the number of animals in the group - 8;

* - statistically significant differences compared with the "Control" group according to the Mann-Whitney test.

2. Influence of the claimed compounds on the behavior of ICR mice under emotional stress in the "elevated cruciform maze" test using the example of compound GD-102.

The Elevated Cruciate Maze (PCL) method is now generally accepted for assessing anxiolytic action (File S.E. Factors controlling measures of anxiety and responses to novelty in the mouse. Behav Brain Res 2001; 125: 151-7). The essence of the method lies in the analysis of the correlation between the fear reaction of animals in an unfamiliar space and height, on the one hand, and search activity in a new environment, on the other.

The PCL installation for mice consists of two intersecting at right angles horizontal tracks 65 × 5 cm.Two opposite compartments have opaque vertical walls 15 cm high.The labyrinth is raised from the floor by 40 cm.At the intersection of the planes there is an open central platform 5 × 5 cm.

The experiment was carried out in daylight conditions. At the beginning of testing, the animal was placed in the center of the maze, which made it possible to move to the dark or light arms of the maze, depending on the prevalence of anxiety (fear of heights, open space) or exploratory activity (which prompted the animal to leave the “protected” arms). The following indicators of behavior in the PCL were recorded for 300 seconds: the time spent in the open arms, the time spent in the closed arms, the time spent on the central site, the number of entries into the open arms, the number of entries into the closed arms.

In the study, outbred male ICR mice weighing 19-25 g (NLP “Nursery of laboratory animals of the FIBC RAS) were used. The animals were kept in a laboratory vivarium under controlled environmental conditions (20-22 ° C and 30-70% relative humidity, 12-hour lighting cycle, 10-fold change in room air volume per hour), in plastic cages with a top cover made of stainless steel with a dust-free bed of wood shavings, 20 mice in each cage, with constant access to extruded briquetted feed GOST R 50258-92 [1993] and drinking water. Animals were divided into groups randomly, according to the criterion of body weight, with a deviation from the mean by no more than ± 10%. Before the experiment, the animals were kept in the experimental room in the "home" cages for 24 hours. The compounds were injected in the form of a suspension with an aqueous solution of Tween-80 (two drops per 10 ml) once intraperitoneally 30 min before the start of the experiment. Statistical processing of the results of pharmacological experiments was performed using one-way analysis of variance ANOVA, Kruskal-Wallis test and non-parametric Wilcoxon-Mann-Whitney U test for independent variables.

Compound GD-102 in the dose range 0.1-0.5 mg / kg demonstrated a significant (p <0.05) anxiolytic effect both in the relative residence time in the open arms and in the relative number of entries into the open arms of the labyrinth (Table 12). ...

Note. The relative residence time in the open arms of the labyrinth was calculated by the formula:

where T ₀ is the residence time of animals in the open arms, T ₃ is the residence time of animals in the closed arms of the labyrinth.

The relative number of entries into the open arms of the labyrinth was calculated using the formula:

where N ₀ is the number of animals entering the open arms, N ₃ is the number of animals entering the closed arms of the labyrinth.

Number of animals in each group n = 8

* p <0.05 - significant differences from the control according to the Mann-Whitney U-test

3. Influence of a specific inhibitor TSPO (18kDa) on the anxiolytic effect of the claimed compounds on the example of compound GD-102

The TSPO (18kDa) receptor is an intracellular protein located on the outer mitochondrial membrane that stimulates steroidogenesis by transporting cholesterol from the outer to the inner mitochondrial membrane. The resulting neurosteroids perform a number of physiological functions, in particular, they are allosteric modulators of the GABA _A receptor, enhancing GABA transmission. Currently, TSPO ligands are considered as promising candidates for the pharmacotherapy of a number of neurological and psychiatric disorders, including anxiety and depression (Rupprecht R., Papadopoulos V., Rammes G, Baghai T.S., Fan J., Akula N., Groyer G ., Adams D., Schumacher M. Translocator protein (18 kDa) (TSPO) as a therapeutic target for neurological and psychiatric disorders. Nat Rev Drug Discov. 2010 Dec; 9 (12): 971-88. Doi: 10.1038 / nrd3295 . Review; Nothdurfter C, Rammes G, Baghai TC, Schüle C, Schumacher M, Papadopoulos V, Rupprecht R. Translocator protein (18 kDa) as a target for novel anxiolytics with a favorable side-effect profile. J Neuroendocrinol. 2012 Jan; 24 (1): 82-92. Doi: 10.1111 / j. 1365-2826.2011.02166.x. Review.).

PK11195 (1- (2-chlorophenyl) -N-methyl- (1-methylpropyl) -3-isoquinoline-carboxamide) is a specific inhibitor that binds to the TSPO receptor site and, when co-administered with TSPO ligands (18 kDa), blocks the chanxiolytic effect (KitaA, KohayakawaH, KinoshitaT, OchiY, NakamichiK, KurumiyaS, FurukawaK, OkaM. Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand. Br J Pharmacol. 2004; 142 (7): 1059-72; Le Fur G , Guilloux F, Rufat P, Benavides J, Uzan A, Renault C, Dubroeucq MC, Guérémy C. Peripheral benzodiazepine binding sites: effect of PK 11195, 1- (2-chlorophenyl) -N-methyl- (1-methylpropyl) - 3 isoquinolinecarboxamide II Invivostudies Life Sci. 1983; 32 (16): 1849-56).

In order to study the dependence of the effect of compound GD-102 on the interaction at the TSPO binding site, the experiment was carried out according to the following scheme: outbred ICR male mice were divided into 4 groups of 8 individuals each. Group 1 was the control, mice were injected intraperitoneally with distilled water at the rate of 0.1 ml per 10 g of body weight, after 30 minutes a repeated similar injection was carried out, and after 30 minutes the behavior of the animals was assessed in the "elevated cruciform maze" test. Mice of group 2 were injected intravenously with PK11195 suspension at a dose of 10.0 mg / kg, after 30 minutes - distilled water, and after 30 minutes the behavior of the animals was assessed in the "elevated cruciform maze" test. Mice of group 3 were injected intravenously with distilled water, after 30 minutes a suspension of GD-23 at a dose of 0.5 mg / kg, and after 30 minutes, the behavior of the animals was assessed in the "elevated cruciform maze" test. Mice of group 4 were injected intravenously with PK11195 suspension at a dose of 10.0 mg / kg, after 30 minutes, the GD-23 suspension at a dose of 0.5 mg / kg, and after 30 minutes, the behavior of the animals was assessed in the "elevated cruciform maze" test.

We used compound GD-102 at a dose of 0.5 mg / kg and PK11195 substance (Sigma-Aldrich, USA) at a dose of 10.0 mg / kg. All substances were prepared in the form of a suspension with Tween-80 and distilled water and were injected with a single intraperitoneal injection.

The experiment was carried out on outbred male ICR mice weighing 19-25 g. The behavior of the animals was assessed in the "elevated cruciform maze" test.

Statistical processing of the obtained results was performed using one-way analysis of variance (ANOVA, Kruskal-Wallis test) and non-parametric analysis for independent variables (Mann-Whitney U-test) (Table 13).

Note. The relative residence time in the open arms of the labyrinth was calculated by the formula:

where T ₀ is the residence time of animals in the open arms, T ₃ is the residence time of animals in the closed arms of the labyrinth.

The relative number of entries into the open arms of the labyrinth was calculated using the formula:

where N ₀ is the residence time of animals in the open arms, N ₃ is the residence time of animals in the closed arms of the labyrinth.

Number of animals in each group n = 8

* p <0.05 - significant differences from the control according to the Mann-Whitney U-test

Compound GD-102 at a dose of 0.5 mg / kg caused a pronounced anxiolytic effect in ICR mice in the PCL test, statistically significantly increasing the time spent in the open arms of the maze and the number of entries into the open arms compared to the control group 1. Preliminary administration of PK11195 at a dose 10.0 mg / kg (group 4) completely blocked the anxiolytic effect of GD-102, the behavior indicators of animals in this group remained at the control level (Table 13).

4. Antidepressant activity of the proposed compounds on the example of compound GD-102 in the forced swimming test according to Porsolt

The antidepressant activity of GD-102 was assessed in the Porsolt forced swimming test [Porsolt RD, Anton G, Blavet N, Jalfre M. Behavioral despair in rats: A new model sensitive to antidepressant treatments. Eur J Pharmacol. 1978; 47 (4): 379-391]. This test is most commonly used worldwide to detect the antidepressant activity of compounds.

On the first day, the mice were placed in cylinders (height 30 cm, diameter 10 cm) filled 2/3 with water at 22 ° C for 10 min. The next day (22 h after the first landing), the animals were again placed in vessels with water under the same conditions, and during the 5-minute test period, the time the animals maintained their characteristic posture of immobility (refusal from active defensive and exploratory behavior) was recorded. The decrease in duration and mice was regarded as evidence of antidepressant activity. In the experiment, male mice of the BALB / c line weighing 19-22 g were used. GD-102 was triturated with Tween80, then diluted with distilled water at the rate of 5 ml / kg and injected into animals at doses of 0.01 and 0.05 mg / kg 60 minutes after the first landing in the cylinders. A 10 mg / kg Amitriptyline solution in distilled water was used as a reference drug. The groups "Control (water)" and "Control (Tween)" were injected intravenously, respectively, with distilled water or distilled water with Tween-80 at the rate of 5 ml / kg.

Experimental groups:

1. "Control (water)" (n = 10)

2. "Control (twin)" (n = 10)

3. "GD-102 (0.01 mg / kg)" (n = 10)

4. "GD-102 (0.05 mg / kg)" (n = 10)

5. "Amitriptyline (10 mg / kg)" (n = 10)

To identify statistical differences between the experimental groups, the Mann-Whitney U test was used.

It was found that in the control groups ("Control (water)" and "Control (twin)"), the total time of immobility was 227 and 239 s, respectively. There were no significant differences between these groups. GD-102 statistically significantly reduced the time of immobility in both studied doses by 9 and 10% compared with the "Control (water)" group and by 15 and 17% compared with the "Control (tween)" group.

Thus, it was shown that compound GD-102 exhibits antidepressant activity at doses of 0.01 and 0.05 mg / kg with a single intraperitoneal injection in the Porsolt forced swimming test on BALB / c mice.

Description of drawings

FIG. 1. The effect of GD-102 with a single intraperitoneal injection on the behavior of BALB / c mice in the "Open field" test with a flash of light, the data are presented as the dependence of the dose of the test compound on the increase in total locomotor activity in relation to the control. Distilled water was used as a control.

The abscissa shows the doses of injected compounds, mg / kg; the ordinate, the proportion of total motor activity relative to the control,%.

* p <0.05 statistically significant differences compared with the "Control" group according to the U-Mann-Whitney test

Claims (26)

1. A dipeptide of the general formula R ₁ -X-AK ₁ Y-AK ₂ -R ₂ , where X = L or D configuration, Y = L or D configuration; AK ₁ = Trp or Gly; AK ₂ = Leu; R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O), or C ₆ H ₅ -CH ₂ -C (O), or CH ₃ - (CH ₂ ) ₄ C (O); R ₂ = OH, or OCH ₃ , or NH ₂ , or NHCH ₃ , which is a translocator protein ligand (TSPO) with neuropsychotropic properties. 2. A dipeptide according to claim 1, characterized in that X = L, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O), R ₂ = NH ₂ . 3. A dipeptide according to claim 1, characterized in that X = D, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O), R ₂ = NH ₂ . 4. The dipeptide according to claim 1, characterized in that X = L, Y = D, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O), R ₂ = NH ₂ . 5. A dipeptide according to claim 1, characterized in that X = L, Y = L, AK ₁ = Gly, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O), R ₂ = NH ₂ . 6. The dipeptide according to claim 1, characterized in that X = L, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O), R ₂ = OCH ₃ . 7. A dipeptide according to claim 1, characterized in that X = L, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O), R ₂ = OH. 8. The dipeptide according to claim 1, characterized in that X = L, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O), R ₂ = NHCH ₃ . 9. A dipeptide according to claim 1, characterized in that X = L, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ -CH ₂ -C (O), R ₂ = NH ₂ . 10. A dipeptide according to claim 1, characterized in that X = L, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = CH ₃ - (CH ₂ ) ₄ -C (O), R ₂ = NH ₂ . 11. A method of treating anxiety conditions by introducing an effective amount of a dipeptide according to claim 1, where X = L, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C ( O), R ₂ = NH ₂ , or X = L, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C (O), R ₂ = OH. 12. A method of treating depressive conditions by introducing an effective amount of a dipeptide according to claim 1, where X = L, Y = L, AK ₁ = Trp, AK ₂ = Leu, R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -C ( O), R ₂ = NH ₂ . 13. A pharmaceutical composition having neuropsychotropic activity, containing as an active substance a therapeutically effective amount of a dipeptide according to claim 1.

Images