Tetrapeptide trp-nle-asp-phenh-ch(ch3)2possessing anxiolytic activity

 

(57) Abstract:

The invention relates to Bioorganic chemistry, namely the synthesis of peptides possessing anxiolytic activity (the ability to control an alarm condition). Describes the new connection tetrapeptide Thr - Nl - s - hN - CH(CH3)2which has a higher anxiolytic activity without side effects, which contributes to the expansion of the means to relieve anxiety. Tetrapeptide can be used as the basis for the development of dosage forms having anxiolytic activity. 5 table.

The invention relates to Bioorganic chemistry, namely the synthesis of peptides possessing anxiolytic activity (the ability to control an alarm condition).

Known peptide - synthetic analogue of the endogenous peptide tuftsin (Thr - Lys - Pro - Arg - Pro - Gly - Pro), peripheral (intraperitoneal) injection in doses 200-3000 mg/kg dosage anxiety in animals with genetically high levels of anxiety (In the journal of Higher nervous activity" name I. P. Pavlova, 1998; 48(1): 153). The disadvantage of this peptide is its low anxiolytic activity (querelle, higher economic costs in its synthesis.

Known peptide - endogenous tetrapeptide cholecystokinin Trp - Met-Asp - PheNH2(SSK-4, or tetragastris) (In the journal European Neuropsychopharmacology.", 1996, V. 6, p. 263). The disadvantage of this tetrapeptide is its entries activity, which limits its use as a drug. With the introduction of tetragastris healthy people in 17% of cases showing signs of anxiety or panic, and in patients with panic condition in 90% of cases registered an increase in the severity of panic symptoms (In the journal "J. Psychiatry-Neuroscience", 1991, V. 16, n 2, p. 91).

The technical result of the invention is to create tetrapeptide Trp - Nle - Asp - PheNH-CH(CH3)2which has a higher anxiolytic activity without side effects, which contributes to the expansion of the means to relieve anxiety.

This result is achieved by the synthesis of tetrapeptide Trp - Nle - Asp - PheNH-CH(CH3)2formula:

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Does not follow from the prior art that the substitution of a methionine residue at the residue of norleucine and C-terminal amide group on isopropylamino in the structure of endogenous CCK-4 will contribute to Nalycheva for drug development, having anxiolytic activity.

This peptide is a white powder, soluble in water with 20% ethanol or acetonitrile, acetic acid, insoluble in hexane, ether. Peptide homogeneous according to thin-layer chromatography, high performance liquid chromatography, characterized by data quantitative amino acid analysis and nuclear magnetic resonance (NMR).

The synthesis of the peptide is carried out by the classical method in the solution by successive growth of the peptide chain by one amino acid, starting from the C-Terminus, using activated esters Z, Boc-amino acids. Lateral carboxyl function aspartic acid defended tert-butilkoi group. Release Boc-, Butprotected tetrapeptide performed with trifluoroacetic acid (TN) with the addition of 5% deionized water and 5% identicial. Purification of the target product is carried out using high performance liquid chromatography (HPLC). Below is an example of obtaining the claimed drug.

In use L-amino acids and their derivatives firms Reanal (Hungary), Bachem, Fluka (Switzerland).

Thin-layer chromatography (TLC) carried out on chromatog 90:10:2 (A); the ethyl acetate - hexane 1:1 (B), chloroform - methanol - 50% acetic acid 85:15:2 (C), chloroform - methanol - 32% acetic acid, 15:4:1 (G). Substances found on the plates using chlorobenzimidazole reagent. Hydrogenation of peptides is carried out in the presence of 10% Pd/C (palladium on coal) company Fluka or Merck (5-10% by weight).

Above the melting temperature (not adjusted) determine on the heating table Boetius (Germany).

N, N-dimethylformamide (DMF) is distilled over ninhydrin and barium oxide, methylene chloride is washed with concentrated sulfuric acid and water, dried over CaCl2, distilled over CaCl2then over calcium hydride.

For extraction from aqueous solutions, crystallization of used solvents brands including H. H., for HPLC acetonitrile (Technofarm, Russia).

Amino acid analysis of peptides, hydrolyzed 6 N. HCl with 2% thioglycolic at 110oC for 24 h, carried out on the automated analyzer Biotronik LC 5001 (Germany).

Analytical HPLC of the target product is performed on the device Gilson (France) in column (4.H mm) Beckman (Ultrasphere ODS) in gradient elution with buffer B (80% acetonitrile + 20% buffer A. buffer A (0.1% aqueous triperoxonane acid). Deth mm).

Solutions are evaporated on a rotary evaporator Buchi (Switzerland) at 40oC.

Z-Phe-NH-CH(CH3)2(I). To a solution of 1.26 g (3 mmol) of Z-Phe-ONp in 10 ml of methanol is added 10 ml of 5% solution of Isopropylamine in chloroform, incubated for 12 h, evaporated to dryness, the product periostat from isopropyl alcohol-hexane. Obtained 1.0 g (96.7%) of compound (I). Rf0.86 (A), 0.94 (D). So pl. 154-155oC.

Z-Asp(OBut)-Phe-NH-CH(CH3)2(II). 0.50 g (1.5 mmol) of the compound (I) hydronaut in 5 ml of a mixture of methanol, ethanol and water in the presence of Pd/C. the Catalyst is filtered off, washed with methanol, the filtrate is evaporated. The residue is dissolved in 5 ml of DMF, the resulting solution was added 0.53 g (1.2 mmol) of Z-Asp(OBut)ONp. After 16 h the reaction mixture was concentrated in vacuo, to the residue are added 50 ml of 5% solution of NaHCO3. Precipitated white precipitate is filtered off, washed on the filter with water until neutral, periostat from isopropyl alcohol-hexane. Obtained 0.60 g (98%) of compound (II). Rf0.89 (A) 0.50 (B).

Z-Nle-Asp(OBut)-Phe-NH-CH(CH3)2(III). 0.60 g of compound (II) dissolved in 5 ml of ethanol and hydronaut similarly, the compound (I). Obtained in the form of oil, the hydrogenation product is dissolved in 5 ml of DMF, the solution was added 0. the mode (III). Rf 0.75 (A) 0.30 (B).

Boc-Trp-Nle-Asp(OBut)-Phe-NH-CH(CH3)2(IV). 0.30 g (0.48 mmol) of the compound (III) hydronaut in ethanol analogously to the compounds (I) and (II). Obtained in the form of oil product was dissolved in 3 ml of DMF, the solution was added 0.21 g (0.5 mmol) of Boc-Trp-ONp. After 16 h, the reaction mixture was treated similarly to the compounds (II) and (III). Obtained 0.28 g (71.8%) of the compound (IV). Rf0.34 (A), 0.57 (B).

Trp-Nle-Asp-Phe-NH-CH(CH3)2(V). 0.16 g (0.21 mmol) of the compound (IV) is dissolved in cooled to 0oC a mixture of 9 ml triperoxonane acid, 0.5 ml of deionized water and 0.5 ml identicial. After 1H the reaction mixture was concentrated in vacuo, the peptide precipitated by cold dry ether. The crude product release is cleaned by the method of preparative HPLC on reversed phase conditions: column diasorb 130 C16T (h mm), gradient elution with buffer B in buffer a - 0.1% of TN, B-80% acetonitrile in a) with a rate of 0.5% per min, flow rate 4 ml/min, detection at 220 nm. Obtained 0.11 g (75%) of compound (V) in the form of a lyophilisate. Rf0.28 (C) 0.55 (D). Rt18.8 min conditions: column Beckman (Ultrasphere ODS 4.6 x 250), gradient elution with buffer B in buffer And 20% to 80% within 30 min (buffer A - 0.1% TN, buffer B was 80% acetonitrile in buffer A). So pl. 132 -140oC.

Example 1. the real weight 100-120 g divided into groups with different levels of endogenous anxiety in test situations of conflict or test Vogel (magazine "Psychopharmacol.", 1971, V. 21, P. 1).

Animal testing in test Vogel, make use of a camera "Lick supression test" firm "Coulbourn" (USA). For 72 hours prior to the experiment, animals deprived of water with an excess of standard briquetted feed, and 24 hours prior to testing, the animals are taught the skill of identifying the source of water. Then in terms of the connection of the electric current (in ascending mode from 0.2 to 1.0 mA) electrode to the cell floor and the drinking water register number approaches an animal to the drinker. The lack of approaches to the drinker with increasing current strength allows us to speak about the increased level of endogenous anxiety in animals. The results of the determinations are given in table.1.

As follows from the results presented in table. 1, the animals of group I is suitable for drinking only when current 0.2 mA, the animals of group II - at 0.2, 0.5 and 1.0 mA, which indicates the high and low levels of endogenous anxiety in animals of groups I and II, respectively.

Then the animals of groups I and II with high and low level alarm accordingly randomly divided into subgroups of 10 rats each. In each group using blind control animals of the same subgroup introduce the claimed connection, and another placebo - 0.9% solution NaC again tested in test Vogel, as described above, by registering the number of approaches to the drinker and the number of shocks received by the animal while the drinkers at an amperage of 0.5 mA. The increase of these parameters in the experimental group of animals compared with the control group receiving 0.9% NaCl solution, is an indicator of the presence of the tested compound anxiolytic activity. The results of the determinations are given in table. 2.

As follows from the results presented in table. 2, in animals with high levels of anxiety, a number of approaches to the drinker and the number of shocks received by the animal during the period of the drinkers was significantly lower than in animals with low levels of anxiety.

With the introduction of the claimed compounds to animals with low level alarm recorded parameters are not changed. With the introduction of the claimed compounds to animals with high levels of anxiety, a number of approaches to the drinker and the number of shocks received during the period of animals drinkers significantly increased compared with the placebo group, and reach values recorded in animals with low levels of anxiety. These results indicate that when the peripheral introduction of the claimed compounds at a dose of 2 µg/kg vasilieuski activity.

Example 2. Having anxiolytic activity in the proposed tetrapeptide evaluate test "raised 4-beam radial maze" (in the book "Techniques and basic experiments for the study of brain and behavior", M,1991). Raised 4-radial maze consists of 4 rays perpendicularly fastened together. The whole installation is raised on a stem at a height of 70 cm from the floor. Each beam has a length of 45 cm width 8 see Two beam darkened by high walls, two remain open, lit.

Experiments are performed on animals with high and low levels of endogenous anxiety. The division of animals carried out by the method described in example 1. Then the animals to the set level alarm randomly divided into subgroups of 10 rats each. Using blind control animals of the same subgroup introduce the claimed connection, and another placebo - 0.9% NaCl solution. The inventive compound is administered intraperitoneally at a dose of 2.0 mg/kg of body weight of the animal for 15 minutes before testing. Next, the animal is placed in the middle and allowed to explore the maze for 5 minutes, recording the following indicators: the number of transitions from one beam to another (I); the number of vyhledavani dark rays (II); number of outputs on OI I, II, III and IV with decreasing index V indicates the reduction of anxiety in animals. The test results are presented in table. 3.

As follows from the results presented in table. 3, in animals with a high level alarm indicators I, II, III and IV below, and the index V is higher than in animals with low levels of anxiety. With the introduction of the claimed compounds to animals with a low level of anxiety, none of the measured characteristics is not changed. With the introduction of the compounds to animals with a high level alarm indicators I, II, III, IV increase and the rate V decreases, reaching the values recorded in animals with low levels of anxiety. These results indicate that when the peripheral introduction of the claimed compounds at a dose of 2 µg/kg of body weight observed relief of anxiety, suggesting that the presence of the compounds anxiolytic activity.

Example 3. In experiment examined the ability of the inventive compounds to modify the density of benzodiazepine receptors in the brain tissue of rats. The density of benzodiazepine receptors (or the number of binding sites of specific ligand - Bmax) is one of the factors in the Kaya pharmacology", 1999, N3, T. 62, page 57). The definition of Bmaxperform using a radioreceptor method.

Experiments are performed on animals with high and low levels of endogenous anxiety. The division of animals carried out in a manner analogous to example 1. Animals with established level of anxiety randomly divided into subgroups of 10 rats each. Using blind control animals of the same subgroup introduce the claimed compound and the other placebo (0.0% NaCl solution). The inventive compound is administered intraperitoneally at a dose of 2 mcg/kg Over 15 minutes after administration of the inventive compounds are slaughtered by decapitation and allocate midbrain. Brain tissue homogenized in 25 volumes of 50 mm Tris-HCl buffer (pH 7,4; 4oC) in the homogenizer of the type of downs. The resulting suspension is centrifuged at 30,000 g for 15 minutes at 4oC. the Supernatant is removed, the residue resuspended in the original volume of 50 mm Tris-HCl buffer (pH 7,4; 4oC) and centrifuged again the centrifugation Procedure was repeated twice.

The reaction mixture of a total volume of 0.5 ml contains: 250 μl of Tris-HCl buffer, 50 µl of labeled ligand, 150 μl of the suspension membrane protein, the final concentration is from 0.2 to 0.8 mg/mlH-diazepam). The binding reaction is conducted for 1 hour at 4oC. Specific interaction of the label with the benzodiazepine receptors is defined as the difference in the number associated with the membranes of radioactivity when the total binding (in the absence of unlabeled ligand) and nonspecific binding (in the presence of 1000-fold excess unlabeled ligand). The reaction is stopped by rapid filtration of the samples through glass fiber filters GF/B (Whatman, England) using machine company Millipore, France. Washed with 6 ml of Tris-HCl buffer, and then dried in air filters placed in vials for scintillation account, fill in 7 ml of dioxane standard scintillator. The radioactivity counted in a scintillation spectrometer RackBeta 1219 (LKB, Sweden) with an efficiency of account at least 30%.

Mathematical processing of the results radioreceptor analysis carried out using the program LIGAND. The results obtained are presented in table. 4.

As follows from the results presented in table. 4, in animals with a high level alarm the number of binding sites 3H-diazepam lower than in animals with a low level. With the introduction of the claimed compounds to animals with a low level of try this figure increases significantly, achieving values recorded in animals with low levels of anxiety. These results indicate that when the peripheral introduction of the claimed compounds at a dose of 2 µg/kg of body weight observed relief of anxiety, suggesting that it has anxiolytic activity. In addition, the presented results demonstrate the involvement of the GABA-benzodiazepine receptor complex in implementation mechanisms of anxiolytic effect of the claimed compounds.

Example 4. In the experiment investigating the effect of the inventive compounds on locomotor activity of animals with high and low levels of endogenous anxiety. For these purposes, the apparatus for monitoring motor activity of small laboratory animals of the firm "Coulbourn" (USA). The division of animals carried out by the method described in example 1. Then the animals to the set level alarm randomly divided into subgroups of 10 rats each. Using blind control animals of the same subgroup introduce the claimed connection, and another placebo - 0.9% NaCl solution. The inventive compound is administered intraperitoneally at a dose of 2.0 mg/kg weight of the animal. After 15 minutes the animal is put in the research to the us in the table. 5.

As follows from the results presented in table. 5, with the introduction of the claimed compounds to animals with high and low level alarm the number of recorded movements of animals does not change. Results indicate no effect of the claimed compounds on locomotor activity of animals with high and low levels of anxiety, which suggests the absence of this compound side effects.

The claimed connection - tetrapeptide Trp - Nle - Asp - PheNH-CH(CH3)2- has advantages over the known peptide compounds, consisting in the presence of higher anxiolytic activity and the absence of side effects in peripheral way of introduction, and the cheapening of the production method, which allows to consider it as a basis for the development of drugs for relief of anxiety.

Tetrapeptide Trp-Nle-Asp-PheNH-CH(CH3)2having anxiolytic activity.

 

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