RU2573823C2 - Substituted dipeptides with neuropsychotropic activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to field of biologically active compounds and relates to novel dipeptides of formula R₁-C(O)-X-Trp-Y-Ile-R₂, where X = L or D configuration, Y = L configuration; R₁ = C₆H₅-CH₂-O, or C₆H₅-(CH₂)₅, or C₆H₅-CH₂; R₂ = OH, or NH₂, or NHCH₃, possessing neuropsychotropic activity, in particular anxiolytic.

EFFECT: obtaining novel biologically active compounds.

9 cl, 10 tbl, 7 ex

Description

Field of Invention

The invention relates to new biologically active dipeptides of the General formula:

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 .

The new compounds are translocation protein ligands (18 kDa, TSPO) and possess neuropsychotropic properties, in particular anxiolytic.

State of the art

Peripheral benzodiazepine receptors (PBR, or TSPO), discovered in 1977 [Braestrup C, Squires R.F., Proc. Natl. Acad. Sci. USA Sep 1977; 74 (9): 3805-3809.], Are located in most peripheral organs, including the kidneys, lungs, heart, but are present in the greatest amount in the tissues producing steroid hormones, such as testicles, ovaries, adrenal glands, and a small amount is present in glial cells of a healthy brain [Beurdeley-Thomas A., Miccoli L., Oudard S., Dutrillaux B., Poupon MF, J. Neurooncol. 2000. V. 46. No. 1 P.45-56]. TSPO is an 18 kDa tryptophan-rich protein that forms a triple protein complex with a 32 kDa voltage-dependent anion channel (VDAC) and 30 kDa adenine nucleotide (ANC) carrier located on the outer mitochondrial membrane [Papadopoulos V., Boujrad N., Ikonomovic MD Ferrara P., Vidic B., Mol. Cell. Endocrinol. 1994. V. 104. N1 P.R5]. In addition, TSPO-associated protein-1 (PRAX-1), which modulates the function of TSPO, is able to bind to this complex. [McEnery M.W., Snowman A.M., Trifdetti R.R., Snyder S.H., Proc. Natl Acad. Sci. USA 1992. V. 89. N8. P.3170.].

TSPO was previously thought to be a subclass of central benzodiazepine receptors since, like them, they bind to diazepam with high affinity. However, the unique structure and location in the cell made TSPO consider a new type of receptor [V. Papadopoulos, M. Baraldi, T.R. Guilarte, T.V. Knudsen, J.-J. Lacapere, P. Lindemann, M.D. Norenberg, D. Nutt, A. Weizman, M.-R. Zhang, M. Gavis, TRENDS in Pharmacological Sciences Vol.27 No.8, P.402-409].

There is abundant evidence of the involvement of TSPO in various biological processes, but its exact physiological role remains unknown. In particular, TSPO is involved in modulating the immune response, porphyrin transport and heme synthesis, regulating cell proliferation, steroid hormone biosynthesis, as well as in programmed cell death [V. Papadopoulos, L. Lecanu, R.C. Brown, Z. Han, Z.-X. Yao, Neuroscience 138 (2006) 749-756]. The most studied function of TSPO is to translate cholesterol from the outer to the inner mitochondrial membrane, which is the limiting stage in the synthesis of steroids and neurosteroids [J.-J. Lacapere, V. Papadopoulos, Steroids 68 (2003) 569-585]. It is well known that neurosteroids play a key role in the functioning of the central nervous system, the formation of mental status, emotions, behavior, learning and memory [Alkayed N.J., Murphy S.J., Traystman R.J., Hurn P.D. J. of the American Heart Asociation. 2000. V.31. P.161-168]. Thus, TSPO can increase the concentration of neurosteroids in the brain, which enhance the neurotransmitter function of GABA and cause non-sedative anxiolytic effects. [G. Primofiore, F. Da Settimo, S. Taliani, F. Simorini, M.P. Patrizi, E. Novellino, G. Greco, E. Abignente, B. Costa, B. Chelli, C. Martini, J. Med. Chem, 2004, v. 47, p. 1852-1855]. There are indications that TSPO ligands may be useful in the treatment of inflammatory and cardiovascular diseases [Roseli C Farges, Sandra R Torresa, Pascual Ferrarab, Rosa M Ribeiro-do-Vallea, Life Sci., 2004, v.74, p.1387 -1395].

TSPO can be used as a target for creating drugs for the treatment of neuronal diseases. Thus, it was shown that TSPO ligands have significant efficacy in various in vivo models of neurodegeneration [Girard, C. et al .Proc. Natl Acad. Sci. USA, 2008, 105, 20505-20510], anxiety [Da Settimo, F. et al. J. Med. Chem. 2008, 51, 5798-5806], depression [Kita A, et al. Br. J. Pharmacol. 2008, 142, 1059-1072]

In the literature, several classes of chemical compounds are described as ligands of TSPO receptors.

As endogenous TSPO agonist ligands, 11 kDa neuropeptide DBI (diazepam binding inhibitor), 16 kDa protein anthralin (anthralin) [Michelle L. James, Silvia Selleri, Michael Kassiou, Curr. Med. Chem., 2006, 13, 1991-2001], StAR (steroidogenic acute regulatory protein) and some porphyrins, including heme [John G. Pastorino, Gabriella Simbula, Ellen Gilfo, J.B. Hoek, and John L. Farber, J. of Biol. Chem., Vol. 269, No.49, Issue of December 9, P.31041-31046].

For a long time, diazepam, clonazepam and some other derivatives of benzodiazepine were considered synthetic TSPO ligands, however, these substances bind to both TSPO and the central benzodiazepine receptor.

A known selective TSPO antagonist is Ro 5-4864 benzodiazepine (application US 20050090489)

The first non-benzodiazepine antagonists of TSPO were the isoquinoline carboxamides PK-1195 and VC-198M [J. Benavides et al, Brain Res. Bull., Vol.13, Iss.1, July 1984, P.69-77].

In 1991, in the patent US 5032595, the company Synthelabo (now Sanofi-Aventis) considered the well-known drug Alpidem as a potential TSPO ligand. It was previously known that this substance has an anxiolytic effect and does not have a sedative effect. The authors of the patent have shown that this compound causes stimulation of steroidogenesis. However, in 1995, Alpidem was withdrawn from sales due to increased hepatotoxicity.

In 1999, imidazopyridine derivatives were described as TSPO ligands in US Pat. No. 5,891,891. The authors have shown their effectiveness in the treatment of extrapyramidal disorders.

In the article [European Journal of Medicinal Chemistry Vol.32, Issue 3, 1997, Pages 241-251], the authors describe benzoxazepines OXA-17f and OXA-17j as TSPO ligands.

In 2007, in the application US 20100179133 as ligands TSPO disclosed substances of General formula

These compounds cause the generation of reactive oxygen species within the mitochondria, which leads to a reduction or prevention of oxidative stress during smoking.

In the international application WO 1999051594, imidazo [1,2-a] pyridines derivatives of the general formula are described:

The authors have shown the effectiveness in the use of the claimed substances as markers of diseases of the central nervous system associated with increased expression of TSPO.

PCT / US2007 / 086538 discloses phenoxyphenyl acetamide primers (DAA-1097, DAA-1106) as biomarkers of molecular imaging of increased TSPO expression.

In the application EP 1036794, Dainippon Pharmaceutical Co., Ltd described a series of 2-aryl-8-oxo-dihydropurines as ligands for TSPO. A study of these compounds showed their activity in a mouse model of Alzheimer's disease (US Pat. No. 6,962,921). Compound XBD-173 (AC-5216, Emapunil), which is N-benzyl-N-ethyl-2- (7-methyl-8-oxo-2-phenylpurin-9-yl) -acetamide, is in the second stage of clinical trials in as an anxiolytic (Novartis, NCT00108836).

The article (Romeo, EA. Et al. J. Pharmacol. Exp. Ter., 1992, 262, 971-978) describes derivatives of arylindoleacetamide that exhibit TSPO agonist activity. Arylindoleacetamide SSR180575 is in the second phase of clinical trials (Sanofi-Aventis, NCT00502515) for the treatment of diabetic neuropathy.

However, the compounds described in the above references do not open up or suggest new structural variations of the patented compounds.

SUMMARY OF THE INVENTION

Our goal was to obtain dipeptide analogues of TSPO ligands with agonistic activity. We have found that this goal can be achieved using compounds of the general formula

R ₁ -C (O) -X-Trp-Y-Ile-R ₂

where X = L or D configuration, Y = L configuration; R ₁ = C ₆ H ₅ —CH ₂ —O—, C ₆ H ₅ - (CH ₂ ) ₅ -, C ₆ H ₅ —CH ₂ -; R ₂ = OH, OCH ₃ , NH ₂ , NHCH ₃

Examples of the invention are the following compounds:

I amide N-carbobenzoxy-L-tryptophanyl-L-isoleucine

N-carbobenzoxy-D-tryptophanyl-L-isoleucine II amide

N-phenylhexanoyl-L-tryptophanyl-L-isoleucine III amide

IV amide N-phenylacetyl-L-tryptophanyl-L-isoleucine

N-carbobenzoxy-L-tryptophanyl-L-isoleucine methyl ester V

VI N-carbobenzoxy-L-tryptophanyl-L-isoleucine

VII N-carbobenzoxy-L-tryptophanyl-L-isoleucine methylamide

Presented in the invention compounds of the invention are further illustrated in table 1.

The above compounds were obtained by well-known methods for the synthesis of peptides. The usual process for the preparation of the subject compounds is the mixing and condensation of the required amino acids, usually in a homogeneous phase.

Condensation in a homogeneous phase can be performed as follows:

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

b) the condensation of an amino acid having an activated carboxyl group and a protected other reactive group with an amino acid that has 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.

A carboxyl group can be activated by converting it to a chlorohydride, azide, anhydride group or an activated ester, such as N-oxysuccinimide, N-hydroxybenzotriazole, pentachlorophenyl or paranitrophenyl ethers. The amino group can be activated by converting it to phosphitamide or the “phosphorus” method.

The most common for the condensation reactions discussed above are: carbodiimide method; azide method; mixed anhydride method; activated ester method. 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.

The preferred condensation methods for the preparation of peptides of formula 1 is the activated ester method, which is carried out mainly using succinimide esters protected by the amino group of amino acids. The best solvents are a mixture of ethyl acetate with dichloromethane, pure ethyl acetate and tetrahydrofuran.

Reactive groups that should not be involved in condensation can be protected by 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 by acid groups, for example, residues of aliphatic, aromatic, heterocyclic carboxylic acids, such as acetyl, benzoyl, pyridinecarboxyl, acid groups, derivatives of carbonic acid, such as ethoxycarbonyl, benzyloxycarbonyl, tert-butyloxycarbonyl; or acid groups derived from sulfonic acids such as para-toluosulfonyl.

In the synthesis of the claimed compounds of N-alkanoyl substituted dipeptides, N-acyl derivatives were prepared using benzyl chloroformate, phenylacetic acid and 6-phenylhexanoic acid. These acyl groups also functioned as N-protecting groups in the subsequent synthesis.

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

Amides of the dipeptides of formula 1 can be obtained by introducing the amide group into the corresponding dipeptide in any suitable way, for example, by treating the dipeptide with ammonia in the presence of a condensing agent. Peptide alkylamides are obtained by aminolysis of the alkyl ester of the corresponding dipeptide or by reaction with the amino acid as the desired alkylamide. Unsubstituted amides, monomethyl and dimethylamides are more preferred.

The peptide esters according to Formula 1 are preferably prepared 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.

Examples of carrying out the invention

In the future, the following abbreviations are used:

Boc - tert-butyloxycarbonyl

DIPEA - Diisopropylethylamine

DMF - dimethylformamide

DMSO - dimethyl sulfoxide

OSu - Oxysuccinyl

Ile - isoleucil

Me - methyl

Ph - phenyl

Phhex - Phenylhexanoyl

TFA - trifluoroacetic acid

THF - tetrahydrofuran

Trp - Tryptophanil

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

Z - benzyloxycarbonyl

TCX - thin layer chromatography

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 ADP 440 Polarimeter automatic polarimeter (Bellingham + Stanley Ltd., England);

¹ H-NMR spectra were recorded on a δ scale, ppm, on a Bruker AC-250 spectrometer (Germany) in DMSO-d ₆ solutions using tetramethylsilane as the internal standard.

The following abbreviations were used to designate resonant signals: s — singlet, d — doublet, t — triplet, k — quadruplet, m — multiplet;

Thin layer chromatography (TCX) was performed on silica gel plates of DC Kieselgel 60 G / F ₂₅₄ (Merck, Germany) in solvent systems chloroform: methanol, 9: 1 (A) or butanol: acetic acid: acid: water 4: 1: 1 (B) , with manifestation in iodine vapor or ultraviolet rays.

EXAMPLE 1. Obtaining the amide N-carbobenzoxy-L-tryptophanyl-L-isoleucine (I), ZL-Trp-L-Ile-NH ₂ .

a) N-tert-butyloxycarbonyl-L-isoleucine, N-Boc-L-Ile.

To a solution of 15.00 g (114.5 mmol) of L-isoleucine in 50 ml of 1N NaOH was added a solution of 10.00 g of NaHCO ₃ in 50 ml of water, 30 ml of isopropyl alcohol and 30.00 g (137.4 mmol, 20% excess) di-tert-butyl pyrocarbonate. The mixture was stirred for 2 hours at room temperature, then diluted with water to a volume of 300 ml. Excess di-tert-butyl pyrocarbonate was extracted with diethyl ether (2 × 100 ml). The aqueous solution was acidified with a 5% solution of H ₂ SO ₄ to pH 4-5, the product was extracted with ethyl acetate (3 × 100). The organic fractions were combined, washed with a saturated NaCl solution, dried over anhydrous MgSO ₄ , the desiccant was filtered off, ethyl acetate was evaporated, and 21.71 g (82%) of pure product were obtained as a colorless transparent oil. The compound was described only by the TCX method (R _f = 0.62 in system A) and was not described by NMR, since additional confirmation of the structure was not required.

b) N-tert-butyloxycarbonyl-L-isoleucine succinimide ester, N-Boc-L-Ile-OSu.

To a solution of 5.00 g (21.6 mmol) of Boc-L-isoleucine in 50 ml of THF was added 2.73 g (23.8 mmol, 10% excess) of N-hydroxysuccinimide, the solution was cooled to 0 ° C and for 30 min., a solution of 4.90 g (23.8 mmol, 10% excess) of dicylohexylcarbodiimide in 20 ml of THF was added dropwise, ensuring that the temperature did not exceed + 5 ° C. Then the reaction mass was stirred for 30 minutes at 0- + 5 ° C and 5 hours at room temperature. The dicyclohexylurea precipitate was filtered off and 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 ° C, the re-formed precipitate of dicyclohexylurea was filtered off, and the filtrate was evaporated. Received a pure product in an amount of 5.31 g (75%) as a light yellow powder with t ° melt. 91.4-93 ° C.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.86 (3 H, t, C ^δ H ₃ Ile), 0.95 (3 H, d, C ^{γ °} H ₃ Ile), 1.29 and 1.47 (2 H, two m, C ^γ H ₂ Ile), 1.38 (9 H, s, -OC (CH ₃ ) ₃ ), 1.86 (1 H, m, C ^β H Ile), 2.79 (4 H, m, OSu), 4.23 (1 H, dd, C ^α H Ile), 7.57 (1 H, d, NH Ile).

c) Amide of N-tert-butoxycarbonyl-L-isoleucine, N-Boc-L-Ile-NH ₂ .

To a solution of 3 g (9 mmol) of Boc-L-Ile-OSu in 5 ml of DMF was added 20 ml of an aqueous solution of ammonia, immediately a white curdish precipitate formed. The resulting suspension was stirred for 30 minutes until the reaction was complete, then it was evaporated to half the volume, 20 ml of chilled distilled water were added. After 2 hours, the precipitate formed was filtered on a glass filter, washed with 20 ml of 3% H ₂ SO ₄ , 30 ml of distilled water, and dried in air. Received 1.94 g (93%) of Boc-Phe-NH ₂ in the form of a white powder with a t ° melt. 167.4-68.2 ° C.

Spectrum ¹ H-NMR (DMSO-d ₆ ) δ, ppm: 0.81 (3 H, t, C ^δ H ₃ Ile), 0.82 (3 H, d, C ^{γ °} H ₃ Ile), 1 , 06 and 1.36 (2 H, two m, C ^γ H ₂ Ile), 1.36 (9 H, s, -OC (CH ₃ ) ₃ ), 1.61 (1 H, m, C ^β H Ile), 2 , 79 (4 H, m, OSu), 3.74 (1 H, dd, C ^α H Ile), 6.53 (1 H, d, NH Ile), 6.97 and 7.25 (2 H, two s, NH ₂ amide )

g) L-isoleucine amide trifluoroacetate, TFA * Ile-NH ₂

To a suspension of 1.30 g (4.9 mmol) of Boc-Ile-NH ₂ in 7 ml of dichloromethane was added 3 ml of TFA and stirred until gas evolution ceased. The solution was evaporated with diethyl ether (3 × 10 ml). The resulting white oil was triturated under diethyl ether. A precipitate was allowed to form for 12 hours in air at room temperature. The crystals were filtered off, dried in a vacuum desiccator over CaCl ₂ . 1.17 g (99%) of TFA * H-Ile-NH _{2 were} obtained as a white powder with a melting point of 135-137 ° C.

Spectrum ¹ H-NMR (DMSO-d ₆ ) δ, ppm: 0.83 (3 H, t, C ^δ H ₃ Ile), 0.91 (3 H, d, C ^{γ °} H ₃ Ile), 1.13 and 1.45 (2 H, two m, C ^γ H ₂ Ile), 1.80 (1 H, m, C ^β H Ile), 3.56 (1 H, dd, C ^α H Ile), 7.55 and 7.82 (2 H, two s, NH ₂ amide), 8.08 (3 H, br.s, N ⁺ H ₃ Ile).

d) 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. Upon cooling, a 50% solution of carbobenzoxychloride in toluene (10 ml, 10% excess) and 100 ml of a 1N NaOH solution were added dropwise from two dropping funnels. The reaction mixture was allowed to warm to room temperature, stirred for 12 hours. At the end of the reaction (TCX control in system B), the alkaline solution was washed with diethyl ether, and the aqueous fraction was neutralized with a 1N HCl solution. The precipitate was filtered off, washed with 0.1N HCl, distilled water, 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 t ° melt. 125-127 ° C.

Spectrum ¹ N-NMR (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, -OCH ₂ C ₆ H ₅ ), 6.87-7.76 (10 N, m, -OCH ₂ C ₆ H ₅ , indole), 7.45 (1 H, d, NH Trp), 10.80 (1 H, s, NH indole).

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

To a solution of 2.00 g (5.9 mmol) of ZL-TrpOH in 15 ml of ethyl acetate (anhydrous, neutral) was added 0.79 g (6.9 mmol, 17% excess) of N-hydroxysuccinimide, the solution was cooled to 0 ° C and for 10 min, then 1.44 g (7.0 mmol, 19% excess) of dicyclohexylcarbodiimide were sprinkled on, making sure that the temperature did not exceed + 5 ° C. The reaction mass was stirred for 30 minutes at a temperature of 0- + 5 ° C and then for 12 hours at room temperature. The dicyclohexylurea precipitate was filtered off and the filtrate was evaporated. The resulting viscous oil was dissolved in 10 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, and the filtrate was evaporated. The resulting oil was washed with hexane, hexane was decanted, the oil was evaporated to dryness. A pure product was obtained in an amount of 2.33 g (92%) as a white foam.

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

g) Amide of N-carbobenzoxy-L-tryptophanyl-L-isoleucine, ZL-Trp-L-Ile-NH ₂ (I).

, c=1, DMF.1.00 g (4 mmol) of TFA * L-IleNH _{2 were} dissolved in 50 ml of a mixture of dichloroethane and ethyl acetate (1: 1) with the addition of 0.8 ml (4 mmol) of DIPEA. With stirring, a solution of 1.96 g (4.4 mmol 10% excess) of ZL-TrpONSu in 20 ml of dichloroethane was added. Stirred for 12 hours at room temperature. At the end of the reaction (TCX control in system A), the solution was extracted with 50 ml of 5% NaHCO ₃ solution and distilled water (2 × 50 ml), the organic phase was evaporated. As evaporation a white precipitate formed. The precipitate was separated on a paper filter, washed with a small amount of methylene chloride, dried in air. The yield was 0.73 g (40%) of pure product with t ° melt. 214-216 ° C, $${\left[\alpha \right]}_{27}^D=-{23}^{\circ }$$

, c = 1, DMF.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.80 (3 H, t, С ^δ Н ₃ Ile), 0.83 (3 H, d, C ^{γ °} Н ₃ Ile), 1.07 and 1.44 (2 H, two m, C ^γ H ₂ Ile), 1.72 (1 H, m, C ^β H Ile), 2.92 and 3.11 (2 H, two dd, C ^β H Trr), 4.17 (1 H d.d., C ^α H Ile), 4.34 (1 H, m, C ^α H Trp), 4.93 and 4.98 (2 H, 2 d, -OCH ₂ C ₆ H ₅ ), 6.95-7.28 (10 H , m, -OCH ₂ C ₆ H ₅ , indole), 7.46 (1 H, d, NH Trp), 7.77 (1 H, d, NH Ile), 7.41 and 7.13 (2 H, two s, NH ₂ amide) ,, 10.80 (1 H, s, NH indole).

EXAMPLE 2. Obtaining the amide N-carbobenzoxy-D-tryptophanyl-L-isoleucine, ZL-Trp-L-Ile-NH ₂ (II)

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

Received similarly ID and IE using D-tryptophan.

b) Amide N-carbobenzoxy-D-tryptophanyl-L-isoleucine, ZD-Trp-L-Ile-NH ₂ (II).

, с=1, DMF.0.50 g (2 mmol) of TFA * L-IleNH _{2 was} dissolved in 50 ml of a mixture of dichloromethane and ethyl acetate (1: 1) with the addition of 0.4 ml (2 mmol) of DIPEA. With stirring, a solution of 0.98 g (2.2 mmol 10% excess) of ZD-TrpOSu in 20 ml of dichloroethane was added. Stirred for 12 hours at room temperature. At the end of the reaction (TCX control in system A), the solution was extracted with 50 ml of 5% NaHCO ₃ solution and distilled water (2 × 50 ml), the organic phase was evaporated. As evaporation a white precipitate formed. The precipitate was separated on a paper filter, washed with a small amount of methylene chloride, dried in air. The yield was 0.80 g (90%) of the product with t ° melt. 195-198 ° C, $${\left[\alpha \right]}_{27}^D=+{\mathrm{thirty}}^{\circ }$$

, c = 1, DMF.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.80 (3 H, t, C ^δ H ₃ Ile), 0.83 (3 H, d, C ^δ H ₃ Ile), 1.07 and 1.44 ( 2 H, two m, C ^γ H ₂ Ilе), 1.72 (1 H, m, C ^β H Ile), 2.92 and 3.11 (2 H, two dd, C ^β H Trp), 4.17 (1 H, dd, C ^α H Ile), 4.34 (1 H, m, C ^α H Trp), 4.93 and 4.98 (2 H, 2 d, -OCH ₂ C ₆ H ₅ ), 6.95-7.28 (10 H, m, -OCH ₂ C ₆ H ₅ , indole), 7.46 (1 H, d, NH Trp), 7.77 (1 H, d, NH Ile), 7.41 and 7.13 (2 H, two s, NH ₂ amide), 10.80 (1 H, s, NH indole).

EXAMPLE 3. Amide N-phenylacetyl-L-tryptophanyl-L-isoleucine, PhCH ₂ C (O) -L-Trp-L-Ile-NH ₂ (III).

a) Synthesis of succinimide ester of phenylacetic acid.

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 for 5 minutes 9.08 g (44.0 mmol, 20% excess) of dicylohexylcarbodiimide were added, ensuring that the temperature did not exceed + 5 ° C. Then the reaction mass was stirred for 30 minutes at a temperature of 0- + 5 ° C and 5 hours at room temperature. The precipitate of dicyclohexylurea 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 the refrigerator at + 8 ° C, the recurring precipitate of dicyclohexylurea was filtered off, the filtrate was evaporated, the oil was washed with hexane, the hexane was decanted, and the residue was evaporated to dryness. A pure product was obtained in an amount of 9.44 g (97%) as a white powder.

Spectrum ¹ H-NMR (DMSO-d ₆ ) δ, ppm: 2.81 (4 H, m, OSu), 4.10 (2 H, s, CH ₂ C ₆ H ₅ ), 7.29-7.42 (5 H, m, C ₆ H ₅ ).

b) L-tryptophanyl-L-isoleucine.

In a flask containing a suspension of 0.10 g of 10% Pd / C in 20 ml of methanol was placed 0.50 g (1.1 mmol) of ZL-Trp-L-IleNH ₂ obtained as in Izh. Hydrogen was passed through the resulting suspension for 3 hours with vigorous stirring. The progress of the reaction was monitored by TCX in the CHCl ₃ : MeOH 9: 1 system. At the end of the reaction, the catalyst was filtered off, washed with methanol and DMF. The filtrate was evaporated to give 0.30 g (85%) of the product as an oil.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.78 (3 H, t, C ^{γ °} Н ₃ Ile), 0.80 (3 H, d, C ^δ H ₃ Ile), 0.95 and 1.34 (2 H, two m, C ^γ H ₂ Ile), 1.64 (1 H, m, C ^β H Ile), 2.75 and 3.11 (2 H, two dd, C ^β H Trp), 3.50 (1 H , m, C ^α H Trp), 4.14 (1 N, dd, C ^α H Ile), 6.94-7.55 (5 H, m, indole), 7.05 and 7.41 (2 H, 2 d, NH ₂ amide ) 7.95 (1 H, d, NH Ile), 10.85 (2 H, br s, NH ₂ Trp).

c) Amide of N-phenylacetyl-L-tryptophanyl-L-isoleucine, PhCH ₂ C (O) -LTrp-L-Ile-NH ₂ (III).

, c=0.5, DMF.To 0.12 g of tryptophanilisoleucine amide in 10 ml of dimethylformamide, 0.10 g of phenylacetic acid succinimide ester was added. The solution was stirred for 24 hours. The progress of the reaction was monitored by TCX method in system A. Excess succinimide ester was quenched by the addition of DMAPA. Then the reaction mass was greatly diluted with distilled water, evaporated on a rotary evaporator. The resulting crystals were transferred to a glass filter, washed with solutions of 3% H ₂ SO ₄ , 5% NaHCO ₃ and distilled water. Dried in a vacuum desiccator over CaCl ₂ , the yield was 0.17 g (98%) of the substance in the form of a white powder with a t ° melt. 226.3-230.0 ° C, $${\left[\alpha \right]}_D^{27}=-{16.2}^{\circ }$$

, c = 0.5, DMF.

Spectrum ¹ H-NMR (DMSO-d ₆ ) δ, ppm: 0.78 (3 H, t, C ^{γ °} Н ₃ Ilе), 0.81 (3 H, d, C ^δ H ₃ Ile), 1.03 and 1.39 (2 H, two m, C ^γ H ₂ Ile), 1.70 (1 H, m, C ^β H Ile), 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, C ^α H Ile), 4.62 (1 H, m, C ^α H Trp), 6.97-7.38 (10 H, 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 Ile), 8.28 (1 H, d, NH Trp), 10.81 ( 1 H, s, NH indole).

EXAMPLE 4. Obtaining the amide N-phenylhexanoyl-L-tryptophanyl-L-isoleucine, Phhex-L-Trp-L-Ile-NH ₂ (IV).

a) Synthesis of 6-phenylhexanoic acid succinimide ester:

5.00 g (26 mmol) of 6-phenylhexanoic acid were dissolved in anhydrous THF. While stirring, 3.30 g (28.6 mmol) of N-hydroxysuccinimide and 5.89 (28.6 mmol) of dicyclohexylcarbodiimide were added. The reaction was carried out for 3 hours at room temperature. The precipitated dicyclohexylurea precipitate was filtered off. The mother liquor was evaporated, diluted with methylene chloride, kept for 12 hours in a refrigerator at + 8 ° C. The re-precipitated precipitate was filtered off, the filtrate was evaporated to dryness. The resulting oil was crystallized in a small amount of isopropyl alcohol. The yield was 5.87 g (76%) in the form of white crystals with t ° pl. 53 ° C.

Spectrum ¹ H-NMR (DMSO-d ₆ ) δ, ppm: 1.39 (2 H, m, CH ₂ chains), 1.62 (4 H, m, (CH ₂ ) ₂ chains), 2 56 (2 H, t, CH ₂ chains), 2.65 (2 H, t, CH ₂ chains), 2.80 (4 N, m, OSu), 7.11-7.32 (5 H, m, -C ₆ H ₅ ).

b) Synthesis of the amide N-phenylhexanoyl-L-tryptophanyl-L-isoleucine Phhex-L-Trp-L-Ile NH ₂ (IV).

, c=0.5, DMF.0.30 g of tryptophanilisoleucine amide, obtained as in IIIb, was dissolved in 5 ml of DMF, and 0.27 g of 6-phenylhexanoic acid succinimide ester was added with stirring. Stirred for 5 hours, then evaporated. The resulting oil was diluted with 50 ml of ethyl acetate, washed with 50 ml of 5% NaHCO ₃ solution, then with distilled water (2 × 50 ml). The organic fraction was separated, dried over MgSO ₄ , and evaporated. Received 0.28 g (60%) of the product in the form of a white powder with t ° melt. 202.1-204.0 ° C, $${\left[\alpha \right]}_D^{27}={130}^{\circ }$$

, c = 0.5, DMF.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.81 (3 H, t, C ^δ H ₃ Ile), 0.84 (3 H, d, C ^{γ °} H ₃ Ile), 1.06 and 1.13 (2 H, two m, C ^γ H ₂ Ile), 1.15 (2 H, t, CH ₂ chains), 1.43 (4 N, m, CH ₂ CH ₂ chains), 1.72 (1 H, m, C ^β N Ile), 2.02 (2 H, t, CH ₂ chains), 2.95 and 3.09 (2 H, two dd, C ^β H Trp), 4.15 (1 H dd, C ^α H Ile 4.59 (1 H m, C ^α H Trp), 6.96-7.60 (10 H, m, C ₆ H ₅ , indole), 7.70 (1 H, d, NH Ile), 8.07 (1 H, d, NH Trp) 7.16 and 7.39 (2 H, two s, NH ₂ amide) ,, 10.80 (1 H, s, NH indole).

EXAMPLE 5. Obtaining methyl ester of L-tryptophanyl-L-isoleucine, L-Trp-L-IleOMe (V).

a) L-isoleucine methyl ester hydrochloride, HCl * L-Ile-OMe.

5.00 g (38 mmol) of L-isoleucine were placed in a three-necked flask equipped with a thermometer, an air cooler and a dropping funnel. 54 ml (133 mmol) of anhydrous methyl alcohol were added (dried over sieves with a pore size of 3 Å). The suspension was cooled to -10 ° C. With stirring, 5.75 ml (80 mmol) of thionyl chloride was added dropwise. The temperature of the reaction mixture was maintained at −10 ° C. during the addition. The reaction was carried out at this temperature until the addition of thionyl chloride was stopped. Then the reaction mixture was warmed to room temperature and left under stirring for 24 hours. Then it was evaporated and placed in a refrigerator for 12 hours. Received glassy crystals of L-isoleucine methyl ester hydrochloride weighing 7.38 g with a pungent odor of almonds, which were dried over KOH and paraffin. Got a pure product weighing 6.65 g (97%).

Spectrum ¹ H-NMR (DMSO-d ₆ ) δ, ppm: 0.86 (3 H, t, C ^δ H ₃ Ile), 0.90 (3 N, d, C ^{γ °} H ₃ Ile) , 1.27 and 1.44 (2 H, two m, C ^γ H ₂ Ile), 1.94 (1 H, m, C ^β H Ile), 3.71 (3 H, s, -OCH ₃ ) , 3.88 (1 N, dd, C ^α H Ile), 8.65 (3 H, br.s, N ⁺ H ₃ Ile)

b) N-carbobenzoxy-L-tryptophanyl-L-isoleucine methyl ester, L-Z-Trp-L-IleOMe (V)

, c=0.5, DMF.1.09 g (6.05 mmol) of HCl * L-Ile-OMe was dissolved in 20 ml of DMF with 1.05 ml (6.05 mmol) of DIPEA, 2.09 g (6.66 mmol) of ZL were added dropwise to this solution. -TrpOSu in 50 ml of DMF. The reaction was conducted for 24 hours at room temperature (TCX control in system A). The reaction mixture was evaporated to dryness, diluted with methylene chloride, the precipitated precipitate of DIPEA chloride was filtered off, the filtrate was washed with 3% H ₂ SO ₄ , then 5% NaHCO ₃ and distilled water. The organic fraction was dried over anhydrous MgSO ₄ , evaporated to dryness, dried in a vacuum desiccator over CaCl ₂ . Received 1,580 g (58%) of the substance in the form of a white foam with t ° melt. 226.3-230.0 ° C, $${\left[\alpha \right]}_D^{27}=-{19.2}^{\circ }$$

, c = 0.5, DMF.

Spectrum ¹ H-NMR (DMSO-d ₆ ) δ, ppm: 0.86 (3 H, d, C ^{γ °} H ₃ Ile), 0.87 (3 N, t, C ^δ H ₃ Ile), 1.20 and 1.41 (2 H, two m, C ^γ H ₂ Ile), 1.80 (1 H, m, C ^β H Ile), 2.90 and 3.06 (2 H, two dd, C ^β H Trp), 3.63 (3 N , s, -OCH ₃ ), 4.29 (1 H, dd, C ^α H Ile), 4.41 (1 H, m, C ^α H Trp),), 4.96 and 5.02 (2 N, 2 d , -OCH 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 Ile ), 10.83 (1H, s, NH indole).

EXAMPLE 6. Obtaining N-carbobenzoxy-L-tryptophanyl-L-isoleucine, Z-LTrp-L-Ile-OH (VI).

a) N-Carbobenzoxy-L-tryptophanyl-L-isoleucine, Z-L-Trp-Ile-OH:

, c=0.5,DMF.1.31 g (2.9 mmol) of ZL-Trp-L-Ile-OMe obtained as in Vb in 10 ml of methanol were dissolved, while a solution of 0.23 g (5.8 mmol) of NaOH in water was poured with stirring. Stirred for 3 hours until complete dissolution of the reaction mass. The reaction mixture was diluted with water, acidified with 10% H ₂ SO ₄ to pH 4. It was extracted with ethyl acetate, the organic phase was dried over anhydrous MgSO ₄ , filtered and re-evaporated 3 times with diethyl ether to obtain a stable foam. The yield was 1.11 g (87%) with t ° melt. 66.4-68.9 ° C, $${\left[\alpha \right]}_D^{27}=-{13.7}^{\circ }$$

, c = 0.5, DMF.

¹ H-NMR spectrum (DMSO-d ₆ ) δ, ppm: 0.77 (3 H, t, C ^{γ °} H ₃ Ile), 0.81 (3 H, d, C ^δ H ₃ Ile), 1.07 and 1.41 (2 H, two m, C ^γ H ₂ Ile), 1.78 (1 H, m, C ^β H Ile), 2.72 and 2.92 (2 H, two dd, C ^β H Trp), 4.02 (1 H d.d., C ^α H Ile), 4.41 (1 H, m, C ^α H Trp),), 4.89 and 4.99 (2 H, s, -OCH ₂ C ₆ H ₅ ), 6.89-7.46 (10 H, m, -OCH ₂ C ₆ H ₅ , indole), 7.65 (1 H, d, NH Trp), 8.07 (1 H, d, NH Ile), 10.84 (1 H, s, NH indole), 12.63 (1 H, broad s, OH).

EXAMPLE 7. Preparation of carbamobenzoxy-L-tryptophanyl-L-isoleucine methylamide, ZL-Trp-L-IleNHCH ₃ (VII).

, c=0.5, DMF.1.05 g (2.3 mmol) of ZL-Trp-L-Ile-OMe, prepared as in Vb, was dissolved in 10 ml of a 5 M alcohol solution of methylamide. Left in a sealed container for 72 hours at room temperature. The precipitate was filtered off, washed with ethyl alcohol, distilled water and diethyl ether. Dried in the air. Received 0.36 g (40%) of the substance in the form of a white powder with t ° melt. 235.6-237.8 ° C, $${\left[\alpha \right]}_D^{27}=-{17.7}^{\circ }$$

, c = 0.5, DMF.

Spectrum ¹ H-NMR (DMSO-d ₆ ) δ, ppm: 0.77 (3 H, t, C ^{γ °} H ₃ Ile), 0.81 (3 N, d, C ^δ H ₃ Ile), 1.16 and 1.39 (2 H, two m, C ^γ H ₂ Ile), 1.68 (1 H, m, C ^β H Ile), 2.57 (3 H, d, -NHCH ₃ ), 2.91 and 3.11 (2 N, two d.d ., C ^β H Trp), 4.12 (1 H, dd, C ^α H Ile), 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 Ile), 10.83 (1 H, s, NH indole).

The results of a pharmacological study of the claimed compounds

1. The effect of compounds GD-23, GD-25, GD-28, GD-30, GD-32, GD-33, GD-36 on the behavior of Balb / c mice under conditions of 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 situation and a threatening situation. The work uses the technique of a lighted “open field”, in which the transfer of an animal from the dark to a brightly lit arena creates an additional stressing factor based on the natural desire of rodents to avoid brightly lit places (SB Seredenin, AA Vedernikov / Effect of psychotropic drugs on the behavior of inbred mice in an open field / Bull. Exp. Biol. Med. - 1979. - T. 88, No. 7, p. 38-40).

The study used male males of the inbred Balb / c line weighing 19-25 g (NPP Nursery of Laboratory Animals of the FIBCH RAS). The animals were kept in 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 dust-free litter 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 average value of no more than ± 10%). Before the experiment, the animals were kept in the experimental room in "home" cages for 24 hours.

Compounds GD-23, GD-25, GD-28, GD-30, GD-32, GD-33, GD-36 were prepared in the form of a suspension with Tween-80 and distilled water and were administered by a single intraperitoneal injection.

30 minutes after intraperitoneal administration of the compound, 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 evenly lit 4 shadowless lamps of 75 W each, located at a height of 1 m above the field surface. The entire arena space 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 curved squares. The animals were monitored for 3 minutes, the number of crossed squares on the periphery (PA), in the central regions (CA), the number of visits to the center (C), as well as the number of vertical struts (VA) and the number of defecations (D) were separately recorded. The total number of crossed squares together with the number of vertical posts was designated as total activity (OA).

The presence of anxiolytic action was judged by the identification of the activating effect on motor activity in animals with the fading reaction in the open field test (Balb / c line).

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

Under conditions of emotional stress in the test "open field" with intraperitoneal administration of compound GD-23 in doses of 0.1; 0.5; and 1.0 mg / kg revealed a statistically significant increase in the peripheral, central and total motor activity of Balb / c mice compared to the control (table 1).

Compounds GD-28 at a dose of 1.0 mg / kg and GD-32 at doses of 1.0 and 5.0 mg / kg statistically significantly (only according to the U-criterion Mann-Whitney) increased peripheral and general motor activity of mice compared with control (table 3 and 5).

Thus, the compound GD-23 in a wide range of doses with the intraperitoneal route of administration has a pronounced anxiolytic effect in Balb / c mice. This activation occurred due to an increase in peripheral and central motor activity, which is typical for compounds with anxiolytic action.

2. The effect of compound GD-23 on the behavior of outbred mice CD-1 in the test "elevated cruciform maze".

The Elevated Cruciform Maze (PCL) method is currently generally accepted for assessing anxiolytic effects (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 is to analyze the ratio of the reaction of fear 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 horizontal tracks 65 × 5 cm that intersect at right angles. Two opposite compartments have opaque vertical walls 15 cm high. The labyrinth is raised 40 cm from the floor. An open central platform of 5 × 5 cm is located at the intersection of the planes.

The experiment was conducted under daylight conditions. At the beginning of testing, the animal was placed in the center of the labyrinth, which made it possible to move into the dark or light arms of the labyrinth, depending on the predominance of anxiety (fear of heights, open space) or research activity (which prompted the animal to leave the “protected” arms). The following indicators of behavior were recorded in PCL for 300 seconds: time spent in open sleeves, time spent in closed sleeves, time spent on the central platform, number of visits to open sleeves, number of visits to closed sleeves.

The study used mongrel male CD-1 mice weighing 19-25 g (NPP Nursery for Laboratory Animals, FIBCH RAS). The animals were kept in 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 dust-free litter 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 average value of no more than ± 10%. Before the experiment, the animals were kept in the experimental room in "home" cages for 24 hours.

Compound GD-23 was prepared as a suspension with tween-80 and distilled water and was administered by a single intraperitoneal injection.

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

In accordance with the characteristics for laboratory mice with behavioral forms, a fear reaction determined the desire of animals to be in the closed arms of the labyrinth, and decreased motor activity. Search (anxiolytic) activity determines the stay of animals in open arms, an increase in motor activity.

Compound GD-23, administered intraperitoneally 30 minutes before the experiment at doses of 0.5 and 1.0 mg / kg, activated the behavior of CD-1 mice (Table 8), statistically significantly reducing the time spent in the closed arms of the labyrinth compared to the control. Thus, in the PKL test, it was shown that compound GD-23 has a significant anxiolytic effect in the dose range of 0.5-1.0 mg / kg.

3. The effect of a specific TSPO inhibitor (18kDa) on the anxiolytic effect of compound GD-23

TSPO (18 kDa), previously designated as the peripheral benzodiazepine 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 GABAA receptor, enhancing the GABA transmission. Currently, TSPO ligands are considered as promising candidates for 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, Schtile C, Schumacher M, Papadopoulos V, Rupprecht R. Translocator protein (18 kDa) as a target for novel anxiolytics with a favored side-effect profile. J Neuroendocrinol. 2012 Jan; 24 (1): 82-92. Doi: 10.1111 / j.1365-2826.2011.02166.x. Review.).

The substance PK11195 (1- (2-chloropheny1) -N-methy1- (1-methylpropyl) -3-isoquinoline-carboxamide) is a specific inhibitor that binds to the TSPO receptor site and, when co-administered with ligands, TSPO (18 kDa) blocks their anxiolytic 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, Gueremy C. Peripheral benzodiazepine binding sites: effect of PK 11195, 1- (2-chloropheny1) -N-methy1- (1- methylpropyl) -3 isoquinolinecarboxamide. II. Invivostudies. LifeSci. 1983; 32 (16): 1849-56).

In order to study the dependence of the effect of the GD-23 compound on the interaction at the TSPO binding site, the experiment was performed as follows: mongrel male CD-1 mice were divided into 4 groups of 8 animals each. Group 1 was a control one, mice were injected intraperitoneally with a dose of 0.1 ml per 10 g of weight, a similar injection was repeated after 30 minutes, and after 30 minutes the animals' behavior was evaluated in the “elevated cross-maze” test. Group II mice were injected with PK11195 suspension at a dose of 10.0 mg / kg, distilled water after 30 minutes, and after 30 minutes the animals' behavior was evaluated in the “elevated cross-maze” test. Distilled water was injected into group 3 mice in br / mice, after 30 min a suspension of GD-23 at a dose of 0.5 mg / kg and after 30 min the behavior of the animals was evaluated in the “elevated cross-maze” test. Group IV mice were injected with a suspension of PK11195 at a dose of 10.0 mg / kg, 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 evaluated in the elevated cross-maze test.

We used the compound GD-23 at a dose of 0.5 mg / kg and the substance RCP 195 (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 administered by a single intraperitoneal injection.

The experiment was performed on outbred mice-males CD-1 weighing 19-25 g. The behavior of the animals was evaluated in the test "elevated cross-shaped labyrinth".

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

Compound GD-23 at a dose of 0.5 mg / kg caused a pronounced anxiolytic effect in CD-1 mice in the PKL test, statistically significantly increasing the time spent in the open arms of the labyrinth and the number of visits to the open arms compared to control group 1. Preliminary administration of PK11195 at a dose of 10.0 mg / kg (Group 4) completely blocked the anxiolytic effect of HD-23, the behavior indicators of animals of this group remained at the control level (Table 9).

The experimental results demonstrate the dependence of the anxiolytic effect of the compound GD-23 on interaction with the TSPO receptor site.

Claims (9)

1. The compounds of General formula R ₁ -C (O) -X-Trp-Y-Ile-R ₂ ,
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 NH _2, or NHCH ₃ having neuropsychotropic activity, provided that the specified compound is not C ₆ H ₅ -CH ₂ -O-C (O) -L-Trp-L-IleNH ₂ . 2. The compound of claim 1,
where X = D, Y = L, R ₁ = C ₆ H ₅ —CH ₂ —O, R ₂ = NH ₂ . 3. The compound of claim 1,
where X = L, Y = L, R ₁ = C ₆ H ₅ -CH ₂ , R ₂ = NH ₂ . 4. The compound according to claim 1,
where X = L, Y = L, R ₁ = C ₆ H ₅ - (CH ₂ ) ₅ , R ₂ = NH ₂ . 5. The compound according to claim 1,
where X = L, Y = L, R ₁ = C ₆ H ₅ —CH ₂ —O, R ₂ = OH. 6. The compound according to claim 1,
where X = L, Y = L, R ₁ = C ₆ H ₅ —CH ₂ —O, R ₂ = NHCH ₃ . 7. The use of the dipeptide amide carbobenzoxy-L-tryptophanyl-L-isoleucine (C ₆ H ₅ -CH ₂ -OC (O) -L-Trp-L-IleNH ₂ ) as an agent with neuropsychotropic activity. 8. A method for treating anxiety and panic conditions by administering an effective amount of a carbobenzoxy-L-tryptophanyl-L-isoleucine amide compound. 9. Pharmaceutical compositions having neuropsychotropic activity, containing as active ingredient a therapeutically effective amount of a compound according to claim 1 or carbobenzoxy-L-tryptophanyl-L-isoleucine amide (C ₆ H ₅ -CH ₂ -OC (O) -L-Trp -L-IleNH ₂ ).