Tetrapeptide trp-nle-asp-phenh-ch(ch3)2with alcohol activity

 

(57) Abstract:

The invention relates to organic chemistry, synthesis of peptides possessing alcohol activity. Describes a new tetrapeptide General formula I Trp-Nle-Asp-PheNH-CH(CH3)2. The technical result of the invention is to create tetrapeptide, which has alcohol activity and has no entries action at peripheral way of introduction, and also the cheaper way to retrieve it. The proposed tetrapeptide can be used as the basis for the development of dosage forms with alcohol activity. 3 table.

The invention relates to organic chemistry, synthesis of peptides possessing alcohol activity.

Known peptide of the formula Asp-Tight(SE)-Met-Gly-Trp-Met - Asp-PheNH2(CCK-8S), with the ability to reduce ethanol consumption in chronically alcoholic animals in its Central (in the ventricles of the brain) introduction (journal of neurochemistry", 1996, No. 2, S. 149). The disadvantages of this peptide are unacceptable Central way of introduction to the practice of medicine, as well as the complexity and high economic costs in its pauseaction) (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)2that has alcohol activity and has no entries action at peripheral way of introduction, and also the cheaper way to retrieve it.

This result is achieved by the synthesis of tetrapeptide formula:

Trp-Nle-Asp-Phe-NH-CH(CH3)2< / BR>
He stems from the prior art that the reduction of the peptide chain and the substitution of the methionine residue at the residue of norleucine and C-terminal amide group on isopropylamino in the structure of endogenous CCK-4 will ensure availability of alcohol activity and lack of entries effect, which makes possible the use of the proposed tetrapeptide as the basis for developing lekarstvennoj, 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 a chromatographic plates Kieselgel 60 (Merck, Germany) in the solvent system: chloroform - methanol - 50% acetic K2% 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.Hm) Beckman (Ultrasphere ODS) in gradient elution with buffer B (80% acetonitrile + 20% buffer A. buffer A (0.1% aqueous triperoxonane acid). The detection is carried out at a wavelength of 220 nm. Preparative HPLC of tetrapeptide spend on column diasorb 130 ST (h mm).

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 NaCO3. 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 0.46 g (1.2 mmol) of Z-Nle-ONp. After 16 h, the reaction mixture was treated similarly to compound (II). Obtained 0.58 g (77%) with the inane (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 1 h 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 ST (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. Rats (male) breed Wistar initial weight 100-120 g is subjected to chronic alcoholism by providing animals for 7 months 10% Rastogi briquetted feed. After 7 months of alcohol abuse animals previously (within 10 days) test for consumption of 10% solution of ethanol while giving the animals water under conditions of free choice, as described in the Methodological recommendations of the Pharmacological Committee of the Ministry of health on experimental (pharmacological) the study of drugs proposed for clinical testing as a means for the treatment and prevention of alcoholism (In the book "Guiding instructional materials for experimental and clinical study of new drugs". Part 3, Moscow, 1981, S. 95. ). After testing, animals are randomly divided into subgroups of 10 rats each. Using blind control animals of one group to impose claimed tetrapeptide, and another placebo - 0.9% NaCl solution. Injection of the drug intraperitoneally, daily for 5 days at doses of 0.5, 1.0, and 2.0 µg/kg of body weight of the animal. Animals are tested for the preferred alcohol consumption, as described above. The results of the determinations conducted using blind controls, are shown in table 1.

As follows from the results presented in table 1, a lower dose of alcohol consumed at the counter"ptx2">

On the contrary, the introduction of tetrapeptide animal model of chronic alcohol intoxication is accompanied by a significant reduction in the dose of alcohol consumed, most pronounced in the introduction of tetrapeptide at a dose of 2.0 mg/kg of body weight.

Example 2. In experiment examined the ability of the proposed tetrapeptide to potentiate the action of narcotic dose of ethanol (4 g/kg of body weight intraperitoneally). The activity indicator connection is changing the lateral position of" rats in the experimental groups compared with the control animals. Experiments are performed on intact and chronically alcoholic animals. Modeling chronic alcohol intoxication carried out in a manner analogous to example 1. The claimed tetrapeptide injected intraperitoneally at a dose of 2.0 mg/kg for 15 minutes prior to the introduction of ethanol. As a placebo, as before, use 0.9% NaCl solution.

The results of the determinations are shown in table 2. As follows from the table 2 data, the claimed tetrapeptide does not change the duration of the "lateral position" in control rats and significantly reduces the duration of the chronic alcoholic rats.

Example 3. In the experiment islenska test: test "bright-dark" camera (In the journal "Pharmacol Biochem Behav", 1980, v. 13, p. 167) and the test of the conflict situation, or test Vogel (magazine "Psychopharmacol.", 1971, v. 21, p. 1).

Before testing in the test "bright-dark" baggage animals within 5 minutes record the locomotor activity. Then the animals are placed in the center of the dark chamber and observed for 5 minutes, recording the time spent in the light chamber (t in light camera in seconds) and number of transitions between dark and light camera. The claimed tetrapeptide injected intraperitoneally 30 min before the test dose of 2 µg/kg of body weight of the animal. Reducing the duration of animals in the light of the camera compared to the control animals treated with 0.9% NaCl solution, is an indicator of the presence of the tested compound entries activity.

Animal testing in test Vogel, make use of a camera "Lick supression test". For 72 hours prior to the experiment, animals deprived of water with an excess of standard briquetted feed. 24 hours before the introduction of the proposed tetrapeptide animals learn the skill of identifying the source of water. Then the animals intraperitoneally injected claimed tetrapeptide at a dose of 2 µg/kg and after 30 minutes under the conditions of connection of electric current strength of 0.5 mA for the electrode M while the drinkers. The reduction 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 entries activity.

The obtained experimental results are presented in table 3.

You can see that with the introduction of the proposed tetrapeptide the time spent by animals in the bright chamber, and the number of approaches to the drinker and the number of shocks received during the period of animals drinkers did not differ from those in the control group. These results indicate that when the peripheral introduction of the proposed tetrapeptide at a dose of 2 mg/kg weight of the studied compounds is not registered entries of the effect.

The claimed tetrapeptide Trp-Nle-Asp-PheNH-CH(CH3)2has advantages over the known peptide compounds, consisting in the presence of alcohol activity and the absence of entries effect in peripheral way of introduction, as well as the cheaper method thereof that allows you to use this connection as the basis for the development of drugs with alcohol activity.



 

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