RU2757476C2 - Leucyltryptophan tspo ligands with anxiolytic activity - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to a new series of biologically active compounds: dipeptides of the general formula: R1-C(O)-L-Leu-L-Trp-R2, where R1is selected from С6Н5-СН2- or C6H5-(CH2)2-; R2is selected from OH, OCN3, NH2or NHCH3.EFFECT: compounds are ligands of the 18 kDa mitochondrial translocator protein (TSPO) and have neuropsychotropic properties, in particular anxiolytic activity.6 cl, 6 dwg, 7 tbl, 7 ex

Description

Scope of invention

The invention relates to a new series of biologically active compounds - dipeptides of the general formula:

R ₁ -C (O) -L-Leu-L-Trp-R ₂ , (1)

where R ₁ = C ₆ H ₅ -CH ₂ - or C ₆ H ₅ - (CH ₂ ) ₂ -;

R ₂ = OH, or OCH ₃ , or NH ₂ , or NHCH ₃ .

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

Epidemiological data indicate that anxiety states, including generalized anxiety, phobic, panic and obsessive-compulsive disorders, occur in 6-10% of the population [International Classification of Diseases (10th revision). Class V: Mental and Behavioral Disorders (F00-F99) (adapted for use in the Russian Federation) .- Rostov-on-Don: Phoenix, 1999.- pp. 245-246.]. Anxiety conditions, comorbid with somatic diseases, aggravate the latter, for example, doubling the mortality rate in cardiovascular diseases. Current drugs for the treatment of anxiety disorders are not effective enough. Therefore, an urgent task is to create new drugs with a wide spectrum of neuropsychotropic activity, including anxiolytic. This problem can be solved by obtaining new ligands of the translocator protein TSPO, which affect the regulation of the synthesis of neurosteroids, modulate GABAergic transmission and thus have a rapid anxiolytic effect. The original TSPO ligands are supposed to be created on the basis of substituted leucyltryptophanes.

State of the art

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 membrane of mitochondria and is responsible for the transfer of cholesterol from the outer membrane to the inner one. This process is the limiting stage of neurosteroid synthesis. In addition, TSPO is involved in the transport of porphyrins, mitochondrial respiration, opening of mitochondrial pores, apoptosis and cell proliferation [Papadopolous V., Baraldi M., Guilarte TR, Knudsen T.V., Lacapere J.-J., Lindemann P. et al ... Translocator protein (18kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol. Sci .. 2006; 27, no. 8: 402-409]. TSPO is found mainly in steroid-generating cells, but is also present in other tissues and organs, such as kidneys, lungs, heart, as well as 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.

The main endogenous ligands of TSPO are cholesterol, porphyrins, and neuropeptides of the endozepin family.

Cholesterol is a key endogenous ligand of TSPO, a precursor for neurosteroid biosynthesis. Cholesterol has a nanomolar affinity for TSPO (Kd <10 nM) [Bordet T, Buisson B, Michaud M, Drouot C, Galea P, Delaage P, et al. Identification and characterization of cholest-4-en-3-one, oxime (TR019622), a novel drug candidate for amyotrophic lateral sclerosis. J. Pharmacol. Exp.Ther 2007; 322 (2): 709-20]. By binding to TSPO, cholesterol is transferred from the outer mitochondrial membrane to the inner one, where under the action of cytochrome P450scc enzymes, 3-β hydroxysteroid dehydrogenase (3β-HSD), 5α-reductase, 3-α-hydroxysteroid dehydrogenase (3α-HSD), which modulate neurosteroid function _A receptor.

Of the porphyrins, protoporphyrin IX (Ki 14.5 ± 10.7 nM), deuteroporphyrin IX (Ki 31.3 ± 2 nM) and heme (Ki 40.6 ± 13.7 nM) have the highest affinity for TSPO [Verma A., Nye J.S., Snyder S.H. Porphyrins are endogenous ligands for the mitochondrial (peripheral-type) benzodiazepine receptor. PNAS 1987; 84 (8): 2256-2260].

The peptide endogenous TSPO ligands include diazepam binding inhibitor (DBI), which is a 9 kDa polypeptide. It was discovered in 1983 in the rat brain while studying proteins that inhibit the binding of benzodiazepine tranquilizers 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 triacontatetroneuropeptide DBI _17-50 (triakontatetraneuropeptide, TTN), also have an affinity for TSPO [Ferrero P., Santiconi MR, Conti-Tron , Costa E., Guidotti A. Study of an octadecaneuropeptide derived from diazepam binding inhibitor (DBI): biological activity and presence in rat brain. PNAS 1986; 83: 827-831].

Derivatives of benzodiazepines, in particular diazepam, are considered the first synthetic ligands of TSPO, however, diazepam itself binds both to TSPO (IC ₅₀ 40 nM) and to the central benzodiazepine receptor (IC ₅₀ 3 nM,) [Braestrup C, Squires RF Specific benzodiazepine receptors in rat brain characterized by high-affinity (3H) diazepam binding. PNAS 1977; 74: 3805-3809]. The classic representative of this group is 4-chlordiazepam (compound Ro5-4864), widely used to study the biological functions of tspo [Gavioli, E., Duaite YS, Bressan E., Ferrara P., Farges, R. C, Monteiro De Lima T C. Antidepressant-like effect of Ro5-4864, a peripheral-type benzodiazepine receptor ligand, in forced swimming test. Eur. J. Pharmacol. 2003; 471: 21-26, Barron AM, Garcia-Segura LM, Caruso D., Jayaraman A., Lee J.-W., Melcangi RC et al. Ligand for translocator protein reverses pathology in a mouse model of Alzheimer's disease. The Journal of Neurosci. 2013; 33 (20): P 8891-8897, Mills C, Makwana ML, Wallace A., Benn S., Schmidt H., Tegeder I. et al. Ro5-4864 promotes neonatal motor neuron survival and nerve regeneration in adult rats. Eur. J. Neurosci. 2008; 27: P.937-946, Leonelli E., Yague JG, Ballabio M., Azcoitia I., Magnaghi V., Schumacher M. et al. Ro5-4864, a synthetic ligand of peripheral benzodiazepine receptor, reduces aging-associated myelin degeneration in the sciatic nerve of male rats. Mechanisms of Aging and Development 2005; 126: 1159-1163.46. Soustiel JF, Zaaroor M., Vlodavsky E., Veenman L., Weizman L., Gavish M. Neuroprotective effect of Ro5-4864 following brain injury. Exp.Neurol. 2008; 214: 201-208, Veiga S., Azcoitia L, Garcia-Segura, LM Ro5-4864, a peripheral benzodiazepine receptor ligand, reduces reactive gliosis and protects hippocampal hilar neurons from kainic acid excitotoxicity. J. Neurosci. Res. 2005; 80: 129-137]

The isoquinoline derivative PK11195, discovered in 1983, was the first non-benzodiazepine compound capable of binding to TSPO with nanomolar affinity (Ki = 9.3 nM) [Le Fur G., Perrier ML., Vaucher K, Imbault F., Flamier A., Benavides J. et al. Peripheral benzodiazepine binding sites: effect of PK 11195, l- (2-chlorophenyl) -N-methyl-N- (l-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 [Tiihonen J. et al. Cerebral benzodiazepine receptor binding and distribution in generalized anxiety disorder: a fractal analysis // Molecular psychiatry. - 1996. - T. 2. - No. 6. - C. 463-471]. Based on the fact that Ro5-4864 is an agonist, it was concluded that PK-11195 is a TSPO antagonist.

A group of scientists from Dainippon Sumitomo Pharma (Japan) has described a group of high-affinity and selective TSPO ligands belonging to the class of benzoxazoloids. The design of the new class was carried out by structural transformation of the benzodiazepine ligand Ro5-4864. In this case, the diazepine ring was opened, and the imine fragment was replaced by an oxazolone heterocycle. The substituents in the formed skeleton were widely varied [Fukaya T., Kodo T., Ishiyama T., Kakuyama N., Nishikawa N., Baba S. et al. Design, synthesis and structure-activity relationships of novel benzoxazolone derivatives as 18 kDa translocator protein (TSPO) ligands. Bioorg. & Med. Chem. 2012; 20: 5568-5582].

Also, Dainippon Sumitomo Pharma (Japan) together with Novartis Pharmaceuticals (Switzerland) created the TSPO ligand, compound AC-5216 (XBD173, Emapunil), an oxopurine derivative that showed anxiolytic and antidepressant effect in Vogel's tests and in the dark-light chamber test [Kita, A .; Kohayakawa, H .; Kinoshita, T .; Ochi, Y .; Nakamichi, K .; Kurumiya, S .; Furukawa, K .; Oka, M. Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand. Br. J. Pharmacol 2004, 142, 1059-1072]. AC-5216 is a Phase II clinical trial by Novartis for the treatment of generalized anxiety disorders (ClinicalTrials.gov identifier: NCT00108836). In this study, AC-5216 showed no difference with placebo in the treatment of anxiety disorders. The lack of activity in stage II may be due to the choice of an inappropriate model.

The authors of the article [Campiani, G .; Nacci, V .; Fiorini, I .; 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, 3435-3450] describe benzoxazepine TSPO ligands capable of competitively displacing PK-11195, of which the OXA-17f compound was the most selective for the TSPO receptor.

In 1990 [Nacci, V .; Fiorini, I .; Garofalo, A .; Cagnotto, A. Farmaco, 1990, 45, 545-557.] A new class of TSPO -benzothiazepine ligands has been proposed. Some compounds of this series, such as THIA-66 and THIA-67, had high affinity and selectivity for TSPO. [Fiorini I., Nacci V., Ciani S. M., Garofalo A., Campiani G., Savini L., Novellino E., Greco G., Bernasconi P., Mennini T.J. A comparative molecular field analysis model for 6-arylpyrrolo [2, l-d] [l, 5] benzothiazepines binding selectively to the mitochondrial benzodiazepine receptor. // J Med Chem. -1994. -№37. -P. 4100-4108.

In the article [Romeo E., Auta J, Kozikowski AP, Ma D, Papadopoulos V, Puia G, Costa E, Guidotti A. 2-Aryl-3-indoleacetamides (FGIN-1): a new class of potent and specific ligands for the mitochondrial DBI receptor (MDR) J. Pharmacol. Exp.Ther., 1992, 262, 971-978] have been described arylindoleacetamide derivatives which are TSPO agonists. Arylindoleacetamide SSR180575 had a high affinity for TSPO (IC ₅₀ 2.5 nM) [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-l-acetamide), a peripheral benzodiazepine receptor ligand, promotes neuronal survival and repair. J Pharmacol Exp Ther. 2002 Jun; 301 (3): 1067-78]. SSR180575 is in the second stage of clinical trials conducted by Sanofi-Aventis (France), (ClinicalTrials.gov identifier: NCT00502515), as a treatment for diabetic neuroiatia.

Synthelabo (now Sanofi-Aventis) during a wide screening of imidazo [1,2-a] pyridine derivatives, in which the anxiolytic, hypnotic, hypnotic and anti-inflammatory properties of these substances were evaluated in animal models in rodents [Kaplan JP., George P. US Patent 4,382,938 A, 1983], a compound named Alpidem was selected. Later it was found that Alpidem has affinity properties for TSPO [Langer S.Z., Arbilla S., Benavides J., Scatton B. Zolpidem and alpidem: two imidazopyridines with selectivity for omega 1- and omega 3-receptor subtypes. Adv. Biochem. Psychopharmacol. 1990; 46: 61-72]. In 1997 the authors of the article [Trapani, G .; Franco, M .; Ricciardi, L .; Latrofa, A .; Genchi, G .; Sanna, E .; Tuveri, P .; Cagetti, E .; Biggio, G .; Liso, G. J. Med. Chem., 1997, 40, 3109-18] based on imidazopyridine derivatives, the compound CB-34 was developed, which had high affinity, selectivity for TSPO and the ability to stimulate steroidogenesis in peripheral tissues.

The authors of the article [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. -№371. -P. 197-204], phenoxyphenylacetamide derivatives, N - (4-chloro-2-phenoxyphenyl) - N - (2-

isopropoxybenzyl) - acetamide, DAA1097 and N- (2,5-dimethoxybenzyl) -N- (5-fluoro-2-phenoxyphenyl) acetamide, DAA1106 as TSPO ligands. These compounds have shown potent anxiolytic effects in in vivo experiments.

In RF patent 2572076, the authors disclosed a number of pyrrolo [1,2- a ] pyrazine derivatives, TSPO ligands, among which the compounds GML-1 and GML-3 had anxiolytic and antidepressant activity in vivo.

The authors of the RF patent 2573823 disclosed TSPO dipeptide ligands of the general formula R ₁ -C (O) -X-Trp-Y-Ile-R2 possessing anxiolytic properties, where 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 . However, the structure of the compounds listed in this description does not imply their new structural variations.

The essence of the invention

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

R ₁ -C (O) -L-Leu-L-Trp-R ₂ ,

where R ₁ = C ₆ H ₅ -CH ₂ - or C ₆ H ₅ - (CH ₂ ) ₂ -; 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-leucyl-L-tryptophan amide

II N-phenylacetyl-L-leucyl-L-tryptophan amide

III methyl ester of L-leucyl-L-tryptophan

IV N-phenylpropionyl-L-leucyl-L-tryptophan

V N-phenylpropionyl-L-leucyl-L-tryptophan methylamide

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

Given in table. 1 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 an acid chloride, azide, anhydride group, or an activated ester such as N-hydroxysuccinimide, N-hydroxybenzotriazole, pentachlophenyl or p-nitrophenyl esters.

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) are the activation of the carboxyl group, which is carried out mainly using succinimide esters of amino group protected amino acids. 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 esterification 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 a sulfonic acid such as p-toluenesulfonyl.

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

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

Amides of dipeptides according to formula (1) are obtained by introducing an amide group into the corresponding dipeptide by treating the activated dipeptide with ammonia. 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.

Examples of implementation of the invention

In what follows, the following abbreviations are used:

Boc - tert-butyloxycarbonyl

DCC - dicyclohexylcarbodiimide

DIPEA - diisopropylethylamine

DMAPA - dimethylpropylene diamine

DMSO - dimethyl sulfoxide

Leu - leucyl

Me - methyl

OSu - hydroxysuccinyl

Ph - phenyl

TFA - trifluoroacetic acid

Trp - tryptophanil

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

Z - benzyloxycarbonyl

DMF - dimethylformamide

DCHM - dicyclohekylurea

THF - tetrahydrofuran

TLC - thin layer chromatography

EA - ethyl acetate

NMR - nuclear magnetic resonance

Starting materials and auxiliary reagents

Amino acids: L-leucine (Alfa Aesar, USA), Z-L-tryptophan (GL-Biochem, Shanghai), L-tryptophan methyl ester hydrochloride (Reanal, Hungary).

Reagents: N-hydroxysuccinimide (Sigma-Aldrich, Germany), dicyclohexylcarbodiimide (Sigma-Aldrich, Germany), di-tert-butylpyrocarbonate (Chemical Line LLC, Russia), trifluoroacetic acid (Khimmed, Russia), Pd / C catalyst (Acros-organics Germany), phenylproionic acid (Alfa Aesar, USA), phenylacetic acid (Alfa Aesar, USA), aqueous ammonia (Khimmed LLC, Russia).

Solvents: Ethyl acetate, dimethylformamide, tetrahydrofuran, diethyl ether, dichloromethane, chloroform, hexane, methanol were obtained from OOO TD Khimmed (Russia). DMF was purified by double distillation in vacuo. The first distillation over calcium hydride, the second distillation over ninhydrin. Tetrahydrofuran was kept over KOH for 24 hours and then distilled at atmospheric pressure. The diethyl ether was kept over CaCl ₂ , then filtered through a paper filter. Dichloromethane and chloroform Was passed through a column with Al ₂ O ₃ . Ethyl acetate and alcohols were used without further purification.

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, Great Britain) in open capillaries without correction;

- the specific rotation of the plane of polarization of light was measured on an automatic polarimeter ADP 440 Polarimeter (Bellingham + Stanley Ltd., Great Britain) at a wavelength of the D line of the sodium spectrum (589.3 nm) and a cuvette length of 1 dm.

Specific optical rotations were calculated using the formula:

[α] _D = (α × V) / (l × a), where

α is the observed optical rotation in degrees; V is the volume of the solution in ml; 1 - layer thickness in dm; a - weight of the substance in g;

- ¹ H-NMR spectra were recorded in the δ, ppm scale, on a spectrometer

Fourier-300 (Bruker, Germany) in DMSO-d ₆ solutions using tetramethylsilane as an internal standard. The following abbreviations were used to designate the resonant signals: s - singlet, d - doublet, 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% alcoholic solution of bromocresol green.

EXAMPLE 1. Obtaining amide N-phenylnropionyl-L-leucyl-L-tryptophan (I), Ph (CH ₂ ) ₂ -C (O) -L-Leu-L-Trp-NH ₂ .

a) 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 ° and then 7.25 g (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 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, OSu), 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, -OCH 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).

b) N-carbobenzoxy-L-tryptophan amide, ZL-Trp-NH ₂ .

ZL-Trp-OSu was dissolved in 10 ml of DMF, 50 ml of aqueous ammonia diluted to 10% and 200 ml of distilled water were added with stirring. The reaction mixture was stirred at room temperature for 0.5 hour, after which it was kept for 3 hours in a refrigerator at + 5 ° C. The precipitate was filtered off, washed with a saturated NaCl solution and distilled water until the washings were neutral. Air dried to constant weight. Received the product in the form of a white powder, yield 90%; m.p. 188-189 ° C, [α] ^D 26 = -27 ° (c 1, DMF). ¹ H-NMR spectrum of DMSO-d ₆ , δ ppm: 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, -OCH ₂ C ₆ H ₅ ), 6.73-7.62 (10 H, m, -OCH ₂ C ₆ H ₅ , indole), 6.89 and 7.19 (2 H, two s, NH ₂ amide), 8.56 (1 H, d, NH Trp), 10.78 (1 H, s, NH indol).

c) HL-tryptophan amide HL-Trp-NH ₂ .

To a solution of 10.80 g (32.0 mmol) of ZL-Trp-NH ₂ in 150 ml of methanol was added 0.59 g of 10% Pd / C (50% humidity) and stirred for 3 h in a hydrogen atmosphere at room temperature. After the disappearance of the starting material (TLC control), the catalyst was filtered off, washed with 100 ml of methanol. The methanol solution was evaporated, 6.17 g (95%) of the product was obtained in the form of an orange oil, [α] ^D 26 = -25 0 (c 1, DMF). ¹ H-NMR spectrum of DMSO-d ₆ , δ ppm: 2.88-3.40 (2 H, br.s, NH ₂ ), 3.17 and 3.39 (2 H, two dd, C ^β H Trp), 3.98 (1 H, m, C ^α H Trp), 6.96-7.34 (10 H, m, indole), 6.98 and 7.15 (2 H, two s, NH ₂ amide), 10.86 (1 H, s, NH indol).

d) N-tert-butyloxycarbonyl-L-leucine, 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. The excess of di-tert-butylpyrocarboate 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, С ^γ H ₂ Leu), 1.36 (9 Н, s, -ОС (СН ₃ ) ₃ ), 1.45 (2 Н, m, С ^β H Leu), 3.86-3.88 (1 H, dd, C ^α H Leu), 6.72 (1 H, d, NH Leu).

e) N-tert-butyloxycarbonyl-L-leucine succinimide ester, 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, the product was obtained in the amount of 8.31 g (73%) in the form of glassy crystals with m.p. 110-112 ° C, [α] _D 26 = -40 ° (c = 2, dioxane). (Ref. Data: mp 116 ° C (diisopropyl ether _{crystal), [α] D} 25 -41.8 ° (c = 2, dioxane) [Anderson, GW; Zimmerman, JE; Callahan, FM The use of esters of N-hydroxysuccinimide in peptide synthesis ../. 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).

f) N-tert-butyloxycarbonyl-L-leucyl-L-trypophan amide, Boc-LLeu-L-Trp-NH ₂ .

To a solution of 3.3 g (16.2 mmol) HL-Trp-NH ₂ in 30 ml DMF was added a solution of 6.00 g (19.4 mmol, 20% eq.) Boc-L-Leu-OSu in 30 ml DMF , stirred for 12 h at room temperature, then 0.2 ml of DMAPA was added and the mixture was stirred for another 30 min. DMF was evaporated, 300 ml of distilled water was added to the still mobile yellow oil, an oily precipitate formed, water over the precipitate was decanted, the residue was dissolved in 250 ml of EA, extracted with 5% NaHCO ₃ (2 × 150 ml) and saturated NaCl solution (1 × 100 ml ). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered, evaporated. The crystals obtained after evaporation were successively washed with distilled water and hexane, and dried in air to constant weight. The product was obtained in the form of cream crystals with a yield of 5.18 g (76%); R _f = 0.48 (A); m.p. 165-166 ° C, [α] ^D 24 = -23 (c 1, DMF). ¹ H-NMR spectrum of 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 Н, m, С ^γ H ₂ Leu), 2.92 and 3.08 (2 Н, two d.d., С ^β H Trp), 1.39 (9 Н, s, -ОС (СН ₃ ) ₃ ), 4.32 (1 Н , dd, C ^α H Leu), 4.61 (1 H, m, C ^α H Trp), 6.91-7.61 (5 H, m, aryl),), 6.98 and 7.15 (2 H, two s, NH ₂ amide), 8.02 (1 H, d, NH Trp), 8.35 (1 H, d, NH Leu), 10.91 (1 H, s, NH indol)

g) L-leucyl-L-trypophan amide trifluoroacetate, TFA * [HL-Leu-L-Trp-NH ₂ ].

To a suspension of 1.50 g (3.6 mmol) of Boc-L-Leu-L-Trp-NH2 in 15 ml of CH ₂ Cl _{2 was} added 4.5 ml of TFA and stirred at room temperature (TLC control in a system (B ), every 1 hour). The reaction was carried out for 3 hours. The reaction mixture was evaporated, after which it was twice pre-evaporated in a mixture with diethyl ether, the still mobile residue was triturated under diethyl ether, the ether was decanted. Received 1.49 g (99%) of the product in the form of a caramel-like substance with so pl. 188-191 ° C, [α] ^D 26 = -25 (c 1, DMF). ¹ H-NMR spectrum of 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), 2.92 and 3.08 (2 H, two dd, C ^β H Trp), 4.32 (1 H, dd, C ^α H Leu), 4.61 ( 1 H, m, C ^α H Trp), 6.91-7.61 (5 H, m, aryl),), 6.98 and 7.15 (2 H, two s., NH ₂ amide), 8.02 (1 H, d, NH Trp), 8.35 (1 H, d, NH Leu), 10.91 (1 H, s, NH indol)

h) Phenylpropionic acid succinimide ester, Ph (CH ₂ ) ₂ -C (O) -OSu

To a solution of 4.00 g (26.66 mmol) of phenylpropionic acid in 100 ml of THF was added 3.68 g (32 mmol) of SuOH, followed by 6.59 g (32 mmol) of DCC. A white precipitate of DCHM was observed to form. The mixture was left under stirring for 12 hours. At the end of the reaction (TLC control in system A, R _f = 0.81), the DCHM precipitate was filtered off, the solvent was removed in a vacuum. The resulting product crystals were dried in an evacuated desiccator over anhydrous Na2SO4. The product yield as a white wax was 6.59 g (97%); R _f 0.90 (A); m.p. 113-114 ° 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, C ₆ H ₅ ).

i) N-phenylpropionyl-L-leucyl-L-tryptophan (I) amide, Ph (CH ₂ ) ₂ C (O) -L-Leu-L-Trp-NH ₂ (GD-136)

To a solution of 1.49 g (3.46 mmol) TFA * [L-Leu-L-Trp-NH ₂ ] in 10 ml of DMF was added 0.63 ml (3.63 mmol, 5% ex.) DIPEA, stirred in within 30 minutes. Then this solution was added to a solution of 1.07 g of Ph (CH ₂ ) ₂ C (O) -OSu (4.32 mmol, 25% g) in 10 ml, stirred for 12 h at room temperature, then 0.1 ml was added N, N-dimethyl-1-aminopropane (DMAPA) and stirred for an additional 30 minutes. DMF was evaporated, 200 ml of distilled water was added to the still mobile yellow oil, and a precipitate formed. The solution with the precipitate was left overnight, then the well-formed precipitate was filtered off, successively washed with 5% NaHCO ₃ , distilled water to pH 7 and hexane, dried in an evacuated desiccator over Na ₂ SO ₄ and paraffin. The product was obtained in the form of cream crystals with a yield of 1.02 g (66%); R _f = 0.41 (A); mp 168-169 ° C, [α] ^D 26 = -15 ° (c 1, DMF). ¹ H-NMR spectrum of 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. Obtaining amide N-phenylacetyl-L-leucyl-L-tryptophan (II), PhCH ₂ -C (O) -L-Leu-L-Trp-NH2 (GD-141).

a) Phenylacetic acid succinimide ester, PhCH ₂ -C (O) -OSu

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. A pure product was obtained in the amount of 9.30 g (90%) in the form of a white powder with mp = 111-112 ° C, R _f 0.89 (A). ¹ H-NMR spectrum (DMSO-d ₆ ) δ, 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) Amide N-phenylacetyl-L-leucyl-L-tryptophaia (II) PhCH ₂ C (O) -L-Leu-L-Trp-NH ₂ (GD-141)

DIPEA (0.63 ml, density 0.742, 5% g) was added to a solution of 1.50 g (3.48 mmol) of TFA [L-Leu-L-Trp-NH ₂ ] obtained as in Ig in 10 ml of DMF, and the mixture was stirred in within 30 minutes. Then this solution was added to a solution of 1.01 g of Ph (CH ₂ ) ₂ C (O) -OSu (4.36 mmol, 25% g) in 10 ml, stirred for 12 h at room temperature, then 0.1 ml was added N, N-dimethyl-1-aminopropane (DMAPA) and stirred for an additional 30 minutes. The mobile oily residue was evaporated off, dissolved in 75 ml of EA, and extracted sequentially with 3% H ₂ SO ₄ (1 × 75 ml), 5% NaHCO ₃ (1 × 75 ml), and distilled water (1 × 75 ml). The organic layer was dried over anhydrous Na2SO4, filtered, evaporated, dried in an evacuated desiccator over Na ₂ SO _4, and paraffin. The product was obtained in the form of a white powder with a yield of 1.12 g (74%); R _f = 0.41 (A); mp 219-221 ° C, [α] ^D 23 = -20 ° (c 1, DMF). ¹ H-NMR spectrum of 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 C ₆ H ₅ ), 4.15 (1 H, dd, С ^α H Leu), 4.62 (1 Н, m, С ^α H Trp), 6.97-7.38 (10 Н, m, СН ₂ С ₆ Н 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 3. Obtaining methyl ester of N-phenylpropionyl-L-leucyl-L-tryptophan (III), Ph (CH ₂ ) ₂ -C (O) -L-Leu-L-Trp-OCH ₃ (GD-138).

a) N-tert-butyloxycarbonyl-L-leucyl-L-trypophan methyl ester, Boc-L-Leu-L-Trp-OCH ₃ .

DIPEA 1.6 ml (10% g) was added to a solution of 2.15 g (8.44 mmol) HCl * [HL-Trp-OCH ₃ ] in 30 ml of DMF, and the mixture was stirred for 30 min. Then a solution of 3.15 g (19.4 mmol, 20% eq.) Of Boc-L-Leu-OSu, obtained as in Ie, in 30 ml of DMF was added, stirred for 12 h at room temperature, then 0.2 ml DMAPA and stirred for an additional 30 minutes. DMF was evaporated, the mobile oily residue was dissolved in 250 ml of EA, extracted sequentially with 3% H ₂ SO ₄ (1 × 100 ml), 5% NaHCO ₃ (1 × 100 ml), and distilled water (1 × 100 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered, evaporated. The caramel-like oil obtained after evaporation was twice re-evaporated with methanol, then triturated with diethyl ether, decanted, and dried in an evacuated desiccator over CaCl ₂ and paraffin to constant weight. The product was obtained in the form of cream crystals with a yield of 3.37 g (76%); R _f = 0.48 (A); m.p. 84-86 ° C, [α] ^D 24 = -23 (c 1, DMF). ¹ H-NMR spectrum of 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), 2.92 and 3.08 (2 H, two d.d., C ^β H Trp), 1.39 (9 H, s, -OC (CH ₃ ) ₃ ), 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 (5 H, m, aryl) , 8.02 (1 H, d, NH Trp), 8.35 (1 H, d, NH Leu), 10.91 (1 H, s, NH indol)

b) Trifluoroacetate methyl ester of NL-leucyl-L-tryptophan TFA * [HL-Leu-L-Trp-OCH ₃ ]

To a suspension of 1.50 g (3.6 mmol) of Boc-L-Leu-L-Trp-OCH ₃ in 15 ml of CH ₂ Cl _{2 was} added 4.5 ml of TFA and stirred at room temperature, after the disappearance of the starting material (TLC- control in system (B), every 1 h), the reaction mixture was evaporated, after which it was twice pre-evaporated in a mixture with diethyl ether, the still mobile residue was triturated under diethyl ether, the ether was decanted. Received 1.49 g (99%) of the product in the form of a creamy powder with so pl. 188-191 ° C, R _f = 0.30 (B), [α] ^D 26 = -25 (c 1, DMF). ¹ H-NMR spectrum of 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), 2.92 and 3.08 (2 H, two dd, C ^β H Trp), 3.61 (3H, 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 (5 H, m, aryl), 8.02 (1 H, d, NH Trp), 8.35 (1 H, d, NH Leu), 10.91 (1 H, s, NH indol)

c) N-phenylpropionyl-L-leucyl-L-tryptophan methyl ester Ph (CH ₂ ) ₂ C (O) -L-Leu-L-Trp-OCH ₃ (GD-138)

To a solution of 2.27 g (5.09 mmol) TFA * [HL-Leu-L-Trp-OCH ₃ ] in 10 ml DMF was added 0.97 ml DIPEA (5.60 mmol, density 0.742, 10% g) , stirred for 30 minutes. Then this solution was added to a solution of _{1.58 g of Ph (CH 2} ) ₂ C (O) -OSu (6.37 mmol, 25% g) in 10 ml, stirred for 12 h at room temperature, then 0.1 ml was added DMAPA and stirred for an additional 30 minutes. The solvent was evaporated, the mobile oily residue was dissolved in 75 ml of EA, and extracted successively with 3% H ₂ SO ₄ (1 × 75 ml), 5% NaHCO ₃ (1 × 75 ml), and distilled water (1 × 75 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered, evaporated. Then the resulting oil was re-evaporated twice with diethyl ether to a foam. Dried in an evacuated desiccator over Na ₂ SO ₄ and paraffin. The product was obtained as a creamy foam with a yield of 2.25 g (93%); m.p. 97-98 ° C; R _f = 0.70 (A); ° C, [α] ^D 26 = -12 ° (c 1, DMF). ¹ H-NMR spectrum of 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 II, 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 4. Obtaining N-phenylpropionyl-L-leucyl-L-tryptophan (IV), Ph (CH ₂ ) ₂ C (O) -L-Leu-L-Trp-OH (GD-139).

Dissolved 1.20 g (2.60 mmol) Ph (CH ₂ ) ₂ C (O) -L-Leu-L-Trp-OCH ₃ , obtained as in IIIc, in 20 ml of MeOH, added with stirring, a solution of 0.21 g (5.20 mmol, 100% eq.) NaOH in 5 ml of water. Stirred for 24 hours. The reaction mixture was diluted with distilled water to a volume of 70 ml, acidified with 5% H ₂ SO ₄ to pH 4, and then transferred to a separatory funnel and extracted with EA (2 × 70 ml). The organic fractions were combined and washed again with saturated NaCl solution. The organic fraction was separated, dried over anhydrous Na ₂ SO ₄ , filtered, evaporated to give a caramel-like product. It was re-evaporated twice with diethyl ether to a foam. Dried in an evacuated desiccator over Na ₂ SO ₄ and paraffin. The product was obtained as a creamy foam with a yield of 1.14 g (97%), m.p. 107-109 ° C; R _f = 0.70 (A); [α] ^D 26 = -8 ° (c 1, DMF). ¹ H-NMR spectrum of 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, OH ).

EXAMPLE 5. Obtaining methylamide N-phenylpropionyl-L-leucyl-L-tryptophan (V), Ph (CH ₂ ) ₂ C (O) -L-Leu-L-Trp-NHCH ₃ (GD-140)

Dissolved 0.42 g (0.91 mmol) Ph (CH ₂ ) ₂ C (O) -L-Trp-L-Leu-OCH ₃ , obtained as in IIIc, in 9 g of a solution of methylamine in ethyl alcohol containing 2, 17 g (91 mmol) of pure methylamine. Left in a sealed container for 6 days at room temperature. The solution was evaporated, then re-evaporated twice with Et2O. Received 0.49 g (96%) of the product in the form of a white powder; m.p. 174 ° C with decomposition, [α] 26 ^D = + 5 °, 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, -NHCH 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, -OCH 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 6. Proof of the ligand properties of the claimed compounds by the method of molecular docking

To confirm the ligand properties with respect to TSPO for compounds GD-136, GD-138, GD-139, GD-140, GD-141, we carried out molecular docking into the active center of TSPO, the structure of which in a complex with the selective ligand PK-11195 obtained by NMR spectroscopy, taken from the Protein Data Bank (PDB identifier 2MGY) [Jaremko L., Jaremko M., Giller K., Becker S., Zweckstetter M. Structure of the mitochondrial translocator protein in complex with a diagnostic ligand. Science, 2014, V343, p. 1363-1366]. Compounds GD-136, GD-138, GD-139, GD-140, GD-141 are localized to the active center of TSPO, while showing high lipophilicity characteristic of all TSPO ligands and binding energy (according to the estimated DS function), comparable to that for selective ligand PK-11195 (see Table 2). For compounds GD-136, GD-139, GD-140, GD-141, the presence of tc-tc stacking between the phenyl fragment of the ligand and the Trp-107 receptor was revealed. The GD-140 compound also exhibits π-π stacking with Trp-95.

A schematic representation of the position of PK-11195, a known selective TSPO ligand, in the active center of TSPO according to docking data is shown in Figure 1. Figures 2-6 show a schematic representation of the positions of GD-136, GD-141, GD-139, GD-140, GD -138 at the TSPO active center according to docking data.

Example 7. 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 with a light flash.

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 distributed 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.

Mice were injected with a suspension of compounds with Tween-80 and distilled water once by intraperitoneal (i.p.) injection. 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 and divided by radii into sectors so that the peripheral ring 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 crossed squares together with the number of upright stands was designated as total activity (TDA), the number of boluses detected during the experiment served as a characteristic of defecation (Def).

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 of compound GD-136 at doses of 0.1-5.0 mg / kg, no statistically significant increase in peripheral, central and general motor activity of mice was found. the BALB / c line compared with the control (Table 3).

The number of animals in the group is 8; Data are presented as S (SD), where S is the mean, SD is the standard deviation; p (ANOVA) - Kruskal-Wallace p-value

Under conditions of emotional stress in the open field test with intraperitoneal administration of compound GD-138 at a dose of 0.1 mg / kg, a statistically significant decrease in peripheral, central, and general motor activity of BALB / c mice was found in comparison with the control (Table 1). 4). When the compound was administered at doses of 0.5 and 5.0 mg / kg, no statistically significant anxiolytic effect was found. When used in a dose of 0.1 mg / kg, a statistically significant increase in the peripheral motor activity of BALB / c mice was found in comparison with the control.

Under conditions of emotional stress in the "open field" test with intraperitoneal administration of compound GD-139 at doses of 0.5 and 5.0 mg / kg, a statistically significant increase in the peripheral, central and general motor activity of BALB / c mice was found in comparison with control (Table 5).

Under conditions of emotional stress in the "open field" test with intraperitoneal administration of compound GD-140 in the dose range of 0.1-1.0 mg / kg, a statistically significant increase in peripheral, central and general motor activity of BALB / c mice was found in comparison with control (Table 6).

Under conditions of emotional stress in the "open field" test with intraperitoneal administration of compound GD-141 in the dose range of 0.1-5.0 mg / kg, a statistically significant increase in the peripheral, central and general motor activity of BALB / c mice was found in comparison with control (Table 7).

Thus, 5 new L-leucyl-L-tryptophan, potential TSPO ligands, were obtained. Anxiolytic activity was revealed for compounds GD-139, GD-140 and GD-141 in the "Open field with a flash of light" test. The most active in this series is the GD-140 compound. Compound GD-138 exhibited anxiogenic activity.

Description of drawings

FIG. 1 Diagram of the position of the RK-11195 in the active center of the TSPO.

FIG. 2 Diagram of the position of the GD-136 in the active center of the TSPO.

FIG. 3 Diagram of the position of the GD-138 in the active center of the TSPO.

FIG. 4 Diagram of GD-139 position in the TSPO active center.

FIG. 5 Diagram of the position of the GD-140 in the active center of the TSPO.

FIG. 6 Diagram of GD-141 position in the TSPO active center.

Claims (13)

1. Compounds with anxiolytic activity of the general formula R ₁ C (O) -L-Leu -L-Trp -R ₂ , where R ₁ can be C ₆ H ₅ -CH ₂ - or C ₆ H ₅ - (CH ₂ ) ₂ -, R ₂ can be —OH or —OCH ₃ or —NH2 or —NHCH ₃ . 2. The connection according to claim 1, characterized in that R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -, R ₂ = -NH ₂ . 3. The connection according to claim 1, characterized in that R ₁ = C ₆ H ₅ -CH ₂ -, R ₂ = -NH ₂ . 4. The connection according to claim 1, characterized in that R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -, R ₂ = -OCH ₃ . 5. The connection according to claim 1, characterized in that R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -, R ₂ = -OH, 6. The connection according to claim 1, characterized in that R ₁ = C ₆ H ₅ - (CH ₂ ) ₂ -, R ₂ = —NHCH ₃ .

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