RU2288736C1 - Peptide possessing anti-coagulant, fibrin-depolymerization, anti-thrombosis and fibrinolytic activities - Google Patents

Abstract

FIELD: medicine, peptides, chemical-pharmaceutical industry.

SUBSTANCE: invention relates to an agent possessing anti-coagulant, fibrin-depolymerization, anti-thrombosis and fibrinolytic activities and comprising peptide of the formula: Lys-Pro-Arg-Pro-Gly-Pro. The claimed agent possesses high activity and effectiveness as compared the known agents.

EFFECT: enhanced medicinal effectiveness of peptide.

1 tbl, 3 ex

Description

The invention relates to medicine, to agents having anticoagulant, fibrindepolymerization, antithrombotic and fibrinolytic activity.

Known heparin with anticoagulant activity (Zaman S.M., Neiring P., Gandhi MR, Gaines PA The pharmacokinetics and US usage of heparin in vascular intervention // Clin.Radiol. 1996. V.51. No. 2. P.113 -116).

However, it has the following disadvantages:

1) does not have antiplatelet properties,

2) does not have fibrinolytic effect,

3) in case of overdose, high molecular fractions can cause undesirable side effects of bleeding.

A known plasmin preparation having a fibrinolytic effect (Gitabert J., Estelles A., Grancella S., Espana F., Aznar J. Fibrinolytic system and reproductive process with special reference to fibrinolytic failure in preeclampsia // Hum. Reprod, 1995. V. 10. Suppl.2. P.121-131).

It has the following disadvantages:

1) does not have anticoagulant effects,

2) does not have antithrombotic action,

3) does not have fibrinopolymerization activity,

4) exhibits a proteolytic effect.

A known preparation of acetylsalicylic acid, which has an antiplatelet effect and weak fibrindepolymerization activity (McKee SA, Sane DC, Deliargyris EN Aspirin resistance in cardiovascular disease: a review of prevalence mechanisms, and clinical significance // Thromb. Haemost. (Schattauer). 2002. V. .88. No. 5. P.711-715).

However, the drug has the following disadvantages:

1) does not detect anticoagulant activity,

2) has side effects - bleeding from the gastrointestinal tract.

Known complex compounds of heparin with blood coagulation proteins - fibrinogen, thrombin. These substances have fibrindepolymerization and antithrombotic activity (Kudrjashov V.A., Lyapina LA Non-enzymatic fibrinolysis and its role in the organism // In book: Thrombosis and thrombolysis. Editors Chazov EI, Smirnov VN / Consultants Bureau (New York). 1986 . P.33-65).

However, these substances have the following disadvantages:

1) have weak anticoagulant activity,

2) do not show antiplatelet effect,

3) the short duration of action in the body,

4) they can not be used in the clinic, as their decay in the bloodstream will increase the level of coagulation factors - fibrinogen, thrombin.

The technical result achieved by the present invention is to expand the spectrum of action of funds on blood coagulation processes.

The specified technical result is achieved by that. that the peptide of the formula:

Lys-Pro-Arg-Pro-Gly-Pro

It is recommended as an agent with anticoagulant, fibrindepolymerization, fibrinolytic and antithrombotic activities.

Example 1. The synthesis of the peptide Lys-Pro-Arg-Pro-Gly-Pro.

The heptapeptide was synthesized by stepwise peptide chain extension using classical methods of peptide chemistry in solution. In the synthesis, the mixed anhydride method and the activated ester method were used. For the synthesis used derivatives of L-amino acids. Evaporation of the solutions was carried out on a rotary evaporator at 40 ° C. Melting points determined on the Boetiys melting table are given without correction. The individuality of the compounds obtained was checked by TLC on silica gel plates from Silufol (Czech Republic). Substances were detected by spraying the plates with a solution of ninhydrin and (or) o-tolidine. The chromatographic mobilities (Rf) in solvent systems are given: acetone: benzene: acetic acid (50: 100: 1) - (1); chloroform: methanol (9: 1) - (2); hexane: acetone (3: 2) - (3); butanol: acetic acid: water (4: 1: 1) - (4); butanol: acetic acid: pyridine: water (30: 6: 20: 24) - (5); chloroform: methanol: ammonia (6: 4: 1) - (6); benzene: ethanol (8: 2) - (7); ethyl acetate: acetone: 50% acetic acid: water (2: 1: 1) - (8); chloroform: methanol (14: 1) - (9): chloroform: methanol: ammonia (8: 1.75: 0.25) - (10); chloroform: methanol: ammonia (6.5: 3.0: 0.5) - (11).

Specific rotation was determined on an A1-EPO polarimeter.

Elemental analysis on the analyzer "Carlo-Erba" model 1106.

Verification of the homogeneity of the peptide was carried out using high performance liquid chromatography. All solvents were absolutated accordingly. Melting points are not corrected.

Accepted abbreviations:

Arg - Arginine

Gly - glycine

Lys - Lysine

Pro - Proline

Boc - tert-butyloxycarbonyl

Z - benzyloxycarbonyl group (KBZ)

OBzl - benzyl ether

OPfp - pentafluorophenyl ether

DMF - dimethylformamide

DHCA - dicyclohexylcarbodiimide

DCM - dicyclohexylurea

THF - tetrahydrofuran

TFA - trifluoroacetic acid

TFA - trifluoroacetate

TEA - triethylamine

EA - ethyl acetate

TLC - thin layer chromatography

PivCl - pivaloyl chloride

I. Preparation of Boc-Pro-Gly-OH.

1. 10.75 g (50 mmol) of Boc-Pro was dissolved in 150 ml of acetonitrile, cooled to -5 ° C, 7.7 ml (50 mmol) of triethylamine (TEA) was added to the solution, cooled to -20 ° C while stirring on a magnetic stirrer . To the cooled solution was added 6.8 ml (55 mmol) of pivaloyl chloride (PivCl), stirred on a magnetic stirrer for 20 minutes at -10 ° C. cooled to −30 ° C. and a pre-cooled Gly solution was added.

A Gly solution was prepared at the same time.

2. Gly 4.5 g (60 mmol, excess 1.2) were dissolved in 35 ml of water and 60 ml of acetonitrile, 8.4 ml (60 mmol) of triethylamine were added, cooled to -10 ° C and after 20 minutes was added to the solution in the first flask. The reaction mixture was kept for 1 hour at -10 ° C and 2 hours at 18-20 ° C, stirring on a magnetic stirrer. The reaction mixture was evaporated on a rotary evaporator. ~ 50 ml of water was added to the residue. The aqueous solution was acidified with a 3-fold excess of NaHSO ₄ (24.84 g) to pH 3, extracted 5 times with 100 ml of ethyl acetate. The combined ethyl acetate solution was washed with H ₂ O (50 ml). 10% KHSO ₄ solution (50 ml), H ₂ O (50 ml), saturated NaCl solution (50 ml). An ethyl acetate solution was dried over MgSO ₄ . Dried ethyl acetate was filtered off and evaporated. Dry ether was added to the residue. When ether was added to the flask, a product precipitated, which was filtered off, washing it with dry ether on the filter. The resulting substance was dried under vacuum in a desiccator over KOH, P ₂ O ₅ and paraffin, changing the desiccants several times.

Yield: 5.97 g (21.74 mmol); (43.5%).

Mp 70 ° C.

Rf 0.863 (1); 0.903 (2); 0.746 (7); 0.847 (8).

II. Getting Boc-Pro-Gly-Pro-OBzl.

1. 5.97 g (21.74 mmol) of Boc-Pro-Gly-OH was dissolved in 100 ml of acetonitrile, cooled to -5 ° C, an excess of 1.1 (3.35 ml; 23.9 mmol) of triethylamine was added to the solution ( TEA), cooled to -20 ° C while stirring on a magnetic stirrer. An excess of 1.1 (2.34 ml; 23.9 mmol) pivaloyl chloride (PivCl) was added to the cooled solution, stirred on a magnetic stirrer for 20 minutes at -10 ° C, cooled to -30 ° C and a pre-cooled solution of HCl · was added Pro-OBzl.

At the same time, a solution of HCl · Pro-OBzl was prepared.

2. HCl · Pro-OBzl 6.3 g (26.1 mmol, excess 1.2) were dissolved in 50 ml of acetonitrile. 4.0 ml (28.71 mmol; excess 1.1) of triethylamine (TEA) were added, cooled to -10 ° C and after 20 minutes was added to the solution in the first flask. The reaction mixture was kept for 1 hour at -10 ° C and 2 hours at 18-20 ° C, stirring on a magnetic stirrer. The reaction mixture was evaporated. 300 ml of ethyl acetate were added to the evaporated residue. The ethyl acetate solution was washed with H ₂ O (3 times 25 ml). 10% KHSO ₄ solution (3 times 25 ml each), H ₂ O (3 times 25 ml each), 5% NaHCO ₃ solution (3 times 25 ml each), N ₂ O (3 times 25 ml each), saturated solution NaCl (1 time 25 ml). An ethyl acetate solution was dried over MgSO ₄ . Dried ethyl acetate was filtered and evaporated. ~ 100 ml of dry ether was added to the residue. When ether was added to the flask, a product precipitated, which was filtered off, washing it with dry ether on the filter. The resulting substance was dried under vacuum in a desiccator over KOH, P ₂ O ₅ and paraffin, changing the desiccants several times.

Yield: 8.12 g (17.66 mmol); (81.23%).

Mp 125-126 ° C.

Rf 0.330 (1); 0.947 (2); 0.390 (3): 0.620 (7).

III. Obtaining TFA-Pro-Gly-Pro-OBzl.

5.358 g (11.65 mmol) of Boc-Pro-Gly-Pro-OBzl was dissolved in 29.13 ml of methylene chloride, 29.13 ml of TFA were added, kept for 45 minutes at room temperature, evaporated twice with abs. ethanol, twice with benzene, twice with ether, was dissolved in benzene and poured with hexane. Hexane was discarded, and the resulting oil was dried in a desiccator over P ₂ O ₅ / KOH and paraffin.

Yield 4.63 g (9.7 mmol) 98%,

Rf - 0.043 (1); 0.247 (2); 0.018 (3),

IV. Preparation of Boc-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl.

3.09 g of Boc-Arg (NO ₂ ) -OH 9.7 mmol was dissolved in 50 ml of THF and 10 ml of DMF, 2.07 g (10.67 mmol) of DHA was added, cooled to 0 ° C, stirred for 40 minutes and a solution of TFA-Pro-Gly-Pro-OBzl in 50 ml of THF and 4.46 ml (9.7 mmol) of TEA was added. The reaction mixture was stirred for three days. The precipitate was filtered with DCM. the solution was evaporated in vacuo, 200 ml of hexane was added to the residue, while the target product was separated in the form of an oil, which was dissolved in 500 ml of ethyl acetate and washed 3 times with 25 ml of 0.1 N HCl. 3 times 25 ml of H ₂ O, 1 time with a saturated solution of NaCl. The organic layer was dried over MgSO ₄ , filtered and evaporated. The residue was dissolved in EA and planted with dry ether. The precipitate was filtered off and dried in vacuo over P ₂ O ₅ / KOH and paraffin, changing the desiccant several times.

Yield 4.76 g (7.9 mmol) 85%.

Mp 108-110 ° C.

Rf 0.44 (4); 0.8 (5).

[α] _D ²² = -77.8 ° (c = 0.5; CH ₃ COOH).

V. Preparation of TFA Arg (NO ₂ ) -Pro-Gly-Pro-OBzl.

4.76 g (7.9 mmol) of Boc-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl was dissolved in 20 ml of methylene chloride, 20 ml of TFA was added. kept for 45 minutes at room temperature, evaporated twice with abs. ethanol. twice with benzene, twice with ether, was dissolved in benzene and poured with hexane. Hexane was discarded, and the resulting oil was dried in a desiccator over P ₂ O ₅ / KOH and paraffin.

The output is quantitative.

Rf 0.16 (4); 0.27 (5).

VI. Obtaining Boc-Pro-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl.

Boc-Pro 1.7 g (7.9 mmol) was dissolved in 20 ml of THF, 1.07 g (7.9 mmol) of MBT was added, cooled to 0 ° C and 1.8 g of DCCA in 50 ml of THF was added. After 40 minutes, a solution of TFA · Arg (NO ₂ ) -Pro-Gly-Pro-OBzl (7.9 mmol) in 50 ml THF and 1.1 ml (7.9 mmol) TEA was added to the reaction mixture. It was stirred for 2 hours at 0 ° C and 2 days at room temperature, then DCM was filtered off, evaporated in vacuo, dissolved in 500 ml of EA and treated similarly with Boc-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl.

Yield 4.07 g (6.5 mmol) of 67.8%.

Mp 147-148 ° C.

Rf 0.42 (4); 0.72 (5); 0.31 (9).

VII. Preparation of TFA · Pro-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl.

Boc-Pro-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl 4.07 g (5.3 mmol) was dissolved in 14 ml of methylene chloride, 14 ml of TFA were added, kept for 45 minutes at room temperature, evaporated twice with abs. ethanol, twice with benzene, twice with ether, was dissolved in benzene and poured with hexane. Hexane was discarded, and the resulting oil was dried in a desiccator over P ₂ O ₅ / KOH and paraffin.

Yield 3.94 g (5.2 mmol) 97%.

Rf 0.246 (2); 0.095 (4): 0.202 (10).

Viii. Preparation of Boc-Lys (Z) -Pro-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl.

1. To Boc-Lys (Z) -OH 3.8 g (10 mmol) in 25 ml abs. EA was added 2.26 g of DCCA and 1.87 g of pentafluorophenol in 50 ml of THF, cooled to 0 ° C. The reaction mixture was stirred for 2 hours at 0 ° C. DCM was filtered off, the solution was evaporated in vacuo, 25 ml of abs was added. ether and 75 ml of hexane, a precipitate gradually separated off upon cooling, which was filtered and dried in vacuo.

Yield 2.8 g (87%).

Mp 117-120 ° C.

Rf 0.84 (4): 0.91 (9).

2. 2.27 g (5.2 mmol) dissolved in 50 ml THF and cooled to 0 ° C were added to TFA · Pro-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl, with stirring, 0.75 ml TEA and then for 2 hours in four portions of Boc-Lys (Z) -OPfp 2.8 g (5.2 mmol). Stirred for 24 hours at room temperature. The reaction mixture was evaporated in vacuo, the residue was dissolved in 300 ml of EA and treated similarly to Boc-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl.

Yield 3 g (3 mmol) 58%.

Mp 130-132 ° C.

Rf 0.38 (4); 0.75 (5).

[α] _D ²² = -86.2 ° (c = 0.5 CH ₃ COOH).

IX. Boc-Lys-Pro-Arg-Pro-Gly-Pro-OBzl.

3 g (3 mmol) of Boc-Lys (Z) -Pro-Arg (NO ₂ ) -Pro-Gly-Pro-OBzl was dissolved in 100 ml of methanol, 0.5 ml of 1N was added. hydrochloric acid and 3 g of catalyst — 10% palladium oxide on neutral alumina, were hydrogenated in a stream of dry hydrogen at room temperature and a pressure of 1 atm for 6 hours. Then the catalyst was filtered off, washed on the filter with methanol. The combined filtrate was evaporated to dryness. The residue was precipitated from abs. methanol dry ether. The precipitate was filtered off, dried in vacuo.

Yield 2.67 g (2.67 mmol) 89%.

X. Lys-Pro-Arg-Pro-Gly-Pro.

2.67 g (2.67 mmol) of Boc-Lys-Pro-Arg-Pro-Gly-Pro-OBzl were suspended in 5 ml of 2 N. hydrochloric acid in dioxane and kept at room temperature for 45 minutes. Then dry ether was added, the precipitate was washed by decantation with dry ether. Periostat from absolute methanol with ether. The resulting precipitate was dissolved in 5 ml of 30% ethanol and applied to a column of Amberlyst A-21 (AcO ^- - form) for exchange to the hydrochloride salt acetate. The peptide was eluted with 100 ml of 30% ethanol, evaporated to dryness in vacuo and precipitated from methanol abs. ether.

Yield 1.54 g (2 mmol) 75%.

Mp - 151 ° C.

Rf 0.24 (6).

[α] _D ²² = -65.0 ° (c = 0.5 CH ₃ COOH).

HPLC Results: Zorbax ODS Column d = 4.6 mm; t = 35 ° C, flow rate 1 ml / min; A = 50 mM NH ₄ H ₂ PO ₄ (pH 2.5); B = A + MeOH (1: 1), 10-60% B (in 25 minutes). Release time 16.5 minutes

Example 2

Take 1 mg of the drug of the formula Lys-Pro-Arg-Pro-Gly-Pro, dissolve in 2.5 ml of physiological saline and administer intravenously in v.jugularis to 5 laboratory rats, 0.5 ml (200 μg) per 200 g of mass body. A control animal was injected with a 0.85% NaCl solution in a dose of 0.5 ml per 200 g of body weight in the same way. After 10 minutes, blood is taken and centrifuged at 2000 rpm for 10 minutes to obtain platelet-poor plasma.

The analysis of the parameters of hemostasis reveals the following. In platelet-poor plasma, a 1.2-fold increase in total fibrinolytic activity was detected, a 1.2-fold increase in anticoagulant activity, a 1.3-fold increase in fibrin-depolymerization activity compared to the control (saline).

Example 3

Take 1 mg of a peptide preparation of the formula Lys-Pro-Arg-Pro-Gly-Pro. dissolved in 2.5 ml of physiological NaCl solution and administered intranasally for 5 days every 24 hours to 5 laboratory rats, 0.5 ml per 200 g of body weight. Control animals in the same way injected 0.85% NaCl solution. 5 days after the last injection, blood was taken from v.jugularis and centrifuged at 2000 rpm for 10 minutes to obtain platelet-poor plasma. Then, in the v.jugularis region (stasis) isolated by clamps, thrombin is caused by thrombin formation and the antithrombotic effect of the drug is determined after 1 hour.

In plasma, an increase in anticoagulant activity was detected - by 1.3 times, total fibrinolytic activity due to an increase in the activity of plasminogen activator - by 1.9 times, fibrin-desolimerization activity -130-160% by 1.5 times compared with the control (saline). The drug showed an antithrombotic effect that exceeded the antithrombotic properties of the blood plasma of healthy rats by 1.45 times: if in the control the blood clot weight was 1.4 mg, then in the experiment it was 0.9 mg.

Thus, the new peptide Lys-Pro-Arg-Pro-Gly-Pro has a wide spectrum of action on the blood coagulation process.

The table shows the evidence of the advantages of the claimed peptide Lys-Pro-Arg-Pro-Gly-Pro compared to the standard preparations of the heparin-fibrinogen complex and tri- and tetra-peptides (Pro-Gly-Pro and Gly-Pro-Gly- Gly).

Table Activities Lys-Pro-Arg-Pro-Gly-Pro Fibrogen-heparin complex (prototype) Pro-Gly-Pro (prototype) Gly-Pro-Gly-Gly (prototype) In vitro
Anticoagulant
130-160%
140%
122-140%
118% Fibrinolytic (total) 30-36 mm ² 30-36 mm ² 36-42 mm ² 24 mm ² Antiplatelet 70% - 83% 75% In vivo Intravenous administration. Anticoagulant activity 120% 135% 173% one hundred% Fibrinolytic non-enzymatic activity 120% 140% 173% one hundred% Tissue plasminogen activator activity 130% 103% 220% one hundred% Platelet Aggregation 108% - 83% one hundred% Intranasal administration Antithrombotic activity (by weight of blood clots) 39% - 19% one hundred% Fibrinolytic total activity 190% - 150% one hundred% Anticoagulant activity 130% - 151% - Fibrindepolymerization Activity 150% - 150% -

Thus, the table shows that the claimed peptide Lys-Pro-Arg-Pro-Gly-Pro has advantages over the preparations of the fibrinogen-heparin complex and the peptides Pro-Gly-Pro and Gly-Pro-Gly-Gly in terms of the increase in anticoagulant, fibrinolytic and antiplatelet activity in vitro; with intravenous administration, the anticoagulant, fibrindepolymerization, fibrinolytic activities of the claimed peptide exceed the same activities of the reference tetrapeptide Gly-Pro-Gly-Gly; when administered intranasally, the antithrombotic effects of the Lys-Pro-Arg-Pro-Gly-Pro peptide exceed that of the Gly-Pro-Gly-Gly tetrapeptide. The fibrinogen-heparin complex is inactive during intranasal administration.

Claims (1)

The peptide of the formula: Lys-Pro-Arg-Pro-Gly-Pro, having anticoagulant, fibrindepolymerization, antithrombotic and fibrinolytic activities.