RU2394836C2 - Peptide having neurotropic activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to bioorganic chemistry and medicine and can be used to prepare medicinal agents with neurotropic activity. The technical result is achieved due to creation of a peptide with neurotropic activity and having general formula: Ac-X-Met-Pro-Arg-Gly-NH₂, where X-Met - L-Met or DL-Met.

EFFECT: obtaining a novel compound - analogues of the C-terminal end of vasopressin which are capable of selectively bonding with different subtypes of vasopressin receptors and, as a result, have different types of neurotropic activity, have low toxicity and are suitable for simple intranasal administration.

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Description

The invention relates to the field of bioorganic chemistry and medicine and can be used to create drugs with neurotropic activity.

It is known that the neurohypophysial hormone vasopressin has behavioral activity, being a stimulant of memory and learning [1, 2]. However, it has a wide spectrum of biological activity and is used primarily in medicine as a hormonal agent that regulates blood pressure and diuresis.

Cystine-containing peptides are known that are fragments of the C-terminal sequence of vasopressin that stimulate learning of laboratory animals at doses 100-1000 times higher than the doses of vasopressin in similar tests, but lacking its hormonal effects [3, 4].

Peptides are known - C-terminal fragments of vasopressin containing cysteine, more accessible synthetically [5, 6]. The most active stimulant of memory and learning among them is the Cys-Pro-Arg-Gly-NH ₂ tetrapeptide, the effective doses of which are comparable to the doses of vasopressin.

Along with peptides of the natural sequence, structural analogues of the C-terminus of vasopressin were obtained. Thus, the replacement of cysteine or cystine with a serine residue led to the hexapeptide pGlu-Asn-Ser-Pro-Arg-Gly-NH ₂ , which is active in tests on the learning ability of laboratory animals at doses 100 times higher than the doses of vasopressin [7]. However, the synthesis of these compounds is complex and multi-stage, since with an increase in the number of amino acid residues, the number of synthesis steps increases.

In the last two decades, several different types of vasopressin receptors have been found and it has been shown that different types of activity are realized when the ligand molecule binds to different types of receptors [8, 9]. Obviously, a narrower spectrum of activity of C-terminal analogues compared to natural hormone is associated with their greater selectivity for vasopressin receptors.

The present invention is the creation of drugs with neurotropic activity.

The technical result that can be obtained by carrying out the claimed invention consists in obtaining new compounds - analogues of the C-terminal region of vasopressin, which can selectively bind to various subtypes of vasopressin receptors and, as a result, possess various types of neurotropic activity (antidepressant effect, stimulating effect on memory processes, motor activity), low toxicity, suitable for administration in an affordable intranasal manner, which can be used for creating nootropic drugs.

This result is achieved by creating a neurotropic activity peptide of the general formula: Ac-X-Met-Pro-Arg-Gly-NH ₂ , where X-Met-L-Met or DL-Met.

To achieve this goal, modifications of the C-terminal vasopressin tetrapeptide were carried out - Cys was replaced with the optical isomers of N-acetylmethionine - Ac-D-Met, Ac-L-Met and Ac-DL-Met. Replacing Cys with Ac-Met stereoisomers allows preserving such important structural elements as the presence of a nucleophilic sulfur atom, and acylation of the α-amino group of methionine increases the enzymatic stability of the peptide. Theoretical conformational analysis showed significant differences in the stable conformations of the obtained stereomers, which leads to high selectivity in binding to various subtypes of vasopressin receptors.

The synthesis of the tetrapeptide Ac-X-Met-Pro-Arg-Gly-NH ₂ , where X-Met-L-Met or DL-Met is carried out using peptide condensation reactions of dicyclohexylcarbodiimide (DCGC) or diisopropylcarbodiimide (DIPC) with the addition of oxybenzotriazole (OBT ) The Boc-Pro-Arg dipeptide is obtained by the method of succinimide esters, then it is condensed with glycine amide, the obtained tripeptides with Boc-protection at the amino group are released by treatment with HCl in ethyl acetate, with the interaction of Pro-Arg-Gly-NH ₂ and Na-acetylated methionine stereomers in the presence of DIPC and MBT produce stereoisomers of peptides of the general formula Ac-X-Met-Pro-Arg-Gly-NH ₂ .

In the developed synthesis schemes, the protection of the guanidine group of arginine is carried out by protonation, which allows one to obtain compounds with a minimum number of synthesis steps due to the elimination of the steps for obtaining N ^G- protected derivatives and cleavage of the protective groups.

A method of synthesizing a peptide of the general formula Ac-X-Met-Pro-Arg-Gly-NH ₂ , wherein X-Met-L-Met or DL-Met is described in the examples. The molecular weight of the synthesized peptides was determined using mass spectrometry. FAB mass spectra were recorded on an LCQ FLEET mass spectrometer ("Thermo"). The homogeneity of the obtained compounds was checked by TLC on Sorbfil plates (Russia), using the following solvent mixtures as eluents: n. Butanol-acetic acid-water, 4: 1: 1 (A); chloroform-methanol - 25% ammonia 60:45:15 (B); chloroform-methanol-25% ammonia-acetic acid 60: 45: 15: 3 (B); ethyl acetate-pyridine-acetic acid-water 10: 10: 3: 6 (D). Substances were detected on chromatograms using a chlorobenzidine sample. The specific rotation of the studied compounds was measured on a Jasco J-20 spectropolarimeter (Japan).

Example 1

N ^α -Tret. Butyloxycarbonyl "prolyl-arginine (Boc-Pro-Arg)

To a solution of 2.15 g (10 mmol) of tert-butyloxycarbonyl-proline and 1.26 g (11 mmol) of N-oxysuccinimide in 12 ml of freshly distilled dioxane was added 2.16 g (10.5 mmol) of DCCA at 7 ° C, intensively stirring the reaction mass. Stirring is carried out for 1 hour while cooling, 3 hours at room temperature. The resulting precipitate of DCMF is filtered off, washed on the filter with 2-3 ml of dioxane. The dioxane solution of Boc-Pro-OSu is added to a solution of 1.22 g (7 mmol) of arginine in 7 ml of water. The reaction mixture is stirred at 20 ° C for 4 h, after the reaction is cooled to 5 ° C, the precipitate of Boc-Pro-Arg is filtered off, washed on the filter with water, cooled to 3-5 ° C (2 × 2 ml), and then ethyl acetate (3 × 3 ml), dried in a vacuum desiccator to constant weight. Yield Boc-Pro-Arg 1.85 g (71%).

Mp 154-155 ° C [α] _D ²⁰ -49 ° C (s 1, methanol), R _f 0.38 (A), 0.71 (B).

Example 2

N ^α -Tert. Butyloxycarbonyl-prolyl-arginyl-glycylamide hydrochloride (Boc-Pro-Arg-Gly-NH ₂ · HCl)

A mixture of 1.11 g (3.0 mmol) of Boc-Pro-Arg and 0.33 g (3.0 mmol) of glycylamide hydrochloride in 7 ml of DMF (the reagents are pre-ground in a mortar to a fine powder) is stirred for 1 h at 20 ° C , cooled to 0-5 ° C and 0.45 g (3.3 mmol) of MBT are added, then 0.68 g (3.3 mmol) of DCCA. The reaction mixture is stirred for 1 hour at (0-5 ° C) and 16 hours at 20 ° C. The precipitate of DCMG is separated by filtration, washed with 2 ml of DMF. 25 ml of ether are added to the combined dimethylformamide solution, after compaction of the precipitate, the solvent layer is decanted, the precipitate is reprecipitated twice from methanol with ether. If there is an impurity of the starting glycinamide (TLC control), the precipitate is dissolved in 3-4 ml of a 1: 1 mixture of ethyl acetate and methanol and ethyl acetate (3-5 ml) is added to the solution in small portions until a precipitate forms. The operation is repeated if the precipitated tripeptide contains an admixture of glycinamide. A homogeneous precipitate according to TLC was dried in vacuo over P ₂ O ₅ .

Yield Boc-Pro-Arg-Gly-NH ₂ · HCl 1.28 g (92%). Mp 85-87 ° C, [α] _d ²⁰ - 51 ° (s 1, methanol), R _f 0.39 (B), 0.51 (D).

Example 3

Prolyl-arginyl-glycylamide dihydrochloride (V) (Pro-Arg-Gly-NH ₂ · 2HCl)

A suspension of 0.93 g (2.0 mmol) of Boc-Pro-Arg-Gly-NH ₂ · HCl in 10 ml of 4.8 N. a solution of HCl in ethyl acetate was stirred for 120 min, controlling the completeness of the removal of the Boc group of TLC in systems (A) or (D), then the solvent was evaporated in vacuo, 5 ml of dry ethyl acetate was added to the residue, and the evaporation operation was repeated. The residue is dissolved in 3 ml of methanol, precipitated by the addition of 12 ml of ether. The precipitate Pro-Arg-Gly-NH ₂ · 2HCl-EA is separated by filtration, washed with ethyl acetate, ether, dried in vacuum over KOH. 0.94 g (96%) of Pro-Arg-Gly-NH ₂ · 2HCl are obtained in the form of a solvate with one molecule of ethyl acetate.

[α] d ²⁰ - 38 ° (s 1, methanol), R _f 0.31 (B), 0.34 (D).

Example 4

N-Acetyl-L-methionyl-prolyl-arginyl-glycylamide (VI) (Ac-L-Met-Pro-Arg-GIy-NH ₂ )

0.135 ml (1.2 mmol) of N-methyl-morpholine are added to a solution of 0.49 g (1.0 mmol) of Pro-Arg-Gly-NH ₂ · 2HCl-EA in 3 ml of DMF cooled to 3 ° C. after 10 minutes, 0.23 g (1.2 mmol) of N-acetyl-D-methionine, 0.16 g (1.2 mmol) of MBT, 0.23 g (1.1 mmol) of DCCH. The reaction mixture was stirred at 3-5 ° C for 1 h and 3 h at 20 ° C, then the precipitate was isolated by filtration with DCMG, washed with 0.5 ml of DMF. 15 ml of ether is added to the combined dimethylformamide solution, after the precipitate is condensed, the supernatant is decanted, the precipitate is dried in vacuo over P ₂ O ₅ , reprecipitated from chloroform with ether and from methanol with ethyl acetate. The precipitate was filtered off and dried in vacuo over P ₂ O ₅ . 0.42 g (83%) of N-Ac-L-Met-Pro-Arg-Gly-NH _{2 is} obtained.

In the presence of impurities of the starting compounds according to TLC, a weighed peptide is dissolved in a minimum amount of methanol and applied to a column of silica gel L 40/100, swollen in eluent chloroform-methanol-water 10: 10: 1. Elution is carried out using the indicated solvent mixture and fractions containing the pure product are collected (TLC control). The combined fractions were evaporated, the residue was reprecipitated from methanol with acetone. After drying over P ₂ O ₅ , pure tetrapeptide is obtained in the form of a white crystalline powder. Yield N-Ac-L-Met-Pro-Arg-Gly-NH ₂ 0.215 g (43%).

Mp 109-110 ° C, [α] _d ²⁰ -57 ° (s 1, methanol),), R _f 0.64 (B), 0.42 (D).

Example 5

N-Acetyl-DL-methionyl-prolyl-arginyl-glycylamide (VI) (Ac-DL-Met-Pro-Arg-Gly-NH ₂ ).

N-Acetyl-DL-methionyl-prolyl-arginyl-glycylamide was prepared according to the procedure described in Example 4 for the L-isomer of 0.49 g (1.0 mmol) of Pro-Arg-Gly-NH ₂ · 2HCl-EA and 0 23 g (1.2 mmol) of N-acetyl-D-methionine. Yield Ac-DL-Met-Pro-Arg-Gly-NH ₂ 0.275 g (55%).

Mp 109-110 ° C, [α] _d ²⁰ -40 ° (s 1, methanol),), R _f 0.64 (B), 0.42 (D).

Experiments to study the effect of · peptides on behavioral reactions were performed on male mongrel white rats weighing 180 ± 30 g. The studied peptides were administered intranasally in an aqueous solution in a volume of 1 μl / 10 g body weight 5 minutes before testing in the study of tentative research behavior, level anxiety and the degree of depression and daily after a training session when developing a conditioned reaction of active avoidance. The animals in the control group were injected with distilled water in an equivalent volume. The drugs were administered in the following doses - 0.01 μg / kg, 0.1, 1.0 and 10.0 μg / kg.

Approximate research behavior of animals was studied in the tests "open field" ("stress-free" and "stressful" modifications) and "mink chamber", the level of anxiety - in the test "raised cross-maze" and "light-dark chamber", the degree of depression - in forced swimming test.

The development of conditions for a one-sided reaction of active avoidance was carried out for four consecutive days.

Ac-L-Met-Pro-Arg-Gly-NI-b when administered intranasally at doses of 1.0-10.0 μg / kg did not affect the orientational-research behavior and the level of anxiety of animals. However, this tetrapeptide had a pronounced antidepressant effect. In the “forced swimming” test, its introduction influenced practically all the studied parameters - a significant increase was recorded both in the duration of the first period of active swimming and in the total time of active swimming, an increase in the total time and average duration of the period of passive swimming; the total time of immobilization and the number of its periods decreased.

Thus, the replacement of the amino acid residue in the tetrapeptide fragment of the vasopressin molecule with L-Met led to the loss of the effect of the peptide on the orientationally-research behavior and the level of anxiety of animals. However, the high level of antidepressant action of Ac-L-Met-Pro-Arg-Gly-NH ₂ indicates, apparently, that the peptide is a selective agonist of V1bR receptors of vasopressin.

The tetrapeptide with a racemic methionine residue Ac-DL-Met-Pro-Arg-Gly-NH ₂ showed a behavioral spectrum similar to the D-isomer (Patent Patent No. 1623166 SU, 15.02.1994 “Neuropeptides with Behavioral Activity”), it stimulated the learning ability of animals in the test of the conditioned reaction of active avoidance increased motor activity and reduced emotional reactivity. In these tests, the effective doses were 5-10 times less compared to the D-isomer and 2 orders of magnitude lower than that of vasopressin.

In addition, he, like the L-isomer, had a pronounced antidepressant effect, which was manifested in doses of 1-10 μg / kg. Thus, Ac-DL-Met-Pro-Arg-Gly-NH ₂ found properties inherent to both the D and L isomers of the tetrapeptide of the general formula Ac-X-Met-Pro-Arg-Gly-NH ₂ , where X-Met - D-Met, L-Met or DL-Met.

Information sources

1. Papsuevich O.S., Chipens G.I., Mikhailova S.V. Neurohypophysial hormones. - Riga: Zinatne. - 1986, 282 p.

2. Walter R., Van Ree J.M., de Wied D. Modification of conditioned benavior of rats by neurohypophysial hormones and analog // Proc. Nat. Acad. Sci USA. - 1978. V.75, N 5. - P.2493-2496.

3. Burbach J.P.H., Leboille J.L.M. Proteolytic conversion of arginine vasopressin and oxytocin by brain synaptic membranes. Characterization of formed peptides and mechanisms of proteolysis // J. Biol. Chem. - 1983. - V.258, N 3. - P.1487-1489.

4. Valter M.P., De Wied D., Van Ree J.M. Vasopressin fragment, AVP (4-9), improves long-term and short-term memory in the hole board search task // Neuropeptides. - 1997. - V.31, N 5. - P.489-494.

5. Golubovich V.P., Martinovich V.P., Usenko A.B. et al. //. Chemical and Pharmaceutical Journal, 1888. - No. 6. - S.675-679.

6. Martinovich V.P., Slobodchikova L.K., Golubovich V.P., Akhrem A.A., Titov S.A., Voskresenskaya O.G. Synthesis and study of the biological activity of the C-terminal fragments of vasopressin - WUA _5-9 and pGlu - WUA _5-9 // Chemical-Pharmaceutical Journal - 1989. - No. 9. - S. 1054-1057.

7. Sato T., Tanaka K., Teramoto T et. Al. Effect of pretraining administration of NC-1900, a vasopressin fragment analog, on memory performance in non- or CO ₂ -amnesic mice Pharmacol. Biochem. Behav. - 2004.V.78. - P.309-317.

8. Lolait S.J., O'Carrol A.M. Brownstein M.J. Molekular biology of vasopressin receptors. Ann. N.Y. Acad. Sci. - 1995 - V.771. P.273-292.

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Claims (1)

A peptide having neurotropic activity of the general formula: Ac-X-Met-Pro-Arg-Gly-NH ₂ ,
where X is Met-L-Met or DL-Met.