SU1083560A1 - Heptapeptide possessing ability of inhibiting myotropic effect of bradikinin - Google Patents

Abstract

Heptapeptide of the formula Val-Tyr-Ile-His-Pro-Phe-Arg with the ability to inhibit the myotropic effect of bradykinin. sl eo co: l 35

Description

one

This invention relates to a new chemical compound, namely to the heptapeptide of the formula

Val-Tyr-Ile-His-Pro-Phe-Arg (1 with the ability to inhibit the myotropic effect of bradykinin, which can be used in experimental biology and medicine.

Bradykinin plays a significant role in inflammatory processes. The biological effects of bradykinin — an increase in vascular permeability, a painful effect, a myotropic effect on smooth muscles, etc. 5 physiologically significant in acute processes, are undesirable in chronic inflammations and in the case of burns.

Known structural analogue of bradykinin - octapeptide formula

835602

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu, which has the ability to inhibit the myotropic effect of bradykinin. However, its biological activity is insufficient to create on its basis medical preparations that inhibit the myotropic effect of bradykinin.

O The purpose of the invention is to expand the arsenal of agents inhibiting the midtropic effect of bradykinin.

The goal is achieved by the heptapeptide formula

15 Val-Tyr-Ile-His-Pro-Phe-Arg

The synthesis of the heptapeptide of formula 1 is carried out by known methods by condensing individual peptide fragments using the azide method according to the scheme below:

Z is benzyloxycarbonyl;

Boc - tert.-butyloxycarbonyl;

Bzl is benzyl;

BzlNOg is p-nitrobenzyl;

NOj nitro group;

Me is methyl;

NP - p-nitrophenyl

In the synthesis were used derivatives of amino acids company Reanal (VNR). All amino acids are L-configuration.

The melting point of the synthesized compounds is determined in open capillaries without correction. The homogeneity of the compounds obtained is monitored by thin layer chromatography (TLC) and NMR. those carried out on plates with Merck silica gel (FRG) with the following systems:

And chloroform - methanol - vinegar acid (85: 10: 5);

B - chloroform - ethanol - n-butanol - ethyl acetate - water (10: 6: 4: 3: 1);

B - chloroform - ethanol - isopropanol - ethyl acetate - water (10: 6: 4: 3: l);

G - chloroform - ethanol - water (40: 47: 5);

D - chloroform - methanol - acetic acid - water (12.0: 41 f6: 6);

E - et1 shaacetate - pyridine - acetic acid - water (5: 3: 1: 2);

W - pyridine - n-butanol - acetic acid - water (10: 15: 3: 12).

Electrophoretic mobility of compounds is given relative to

to histidine (E „, g on paper FN -15 in 5 n (pH 1.9) acetic acid.

The amino acid composition was determined on a Liquimat III analyzer from Kontron (Germany).

An example. Getting heptapeptide formula.

1. Benzyloxycarbonyl-valyl-O-benzyl-tyrosyl isoleate-N-benzyl-histidyl-proline Z-V methyl ester

-Tyr (Bzl) -Ile-His (Bzl) -Pro-Ome (ll)

To cooled to a solution of 20.74 g (40 mmol) of benzyloxycarbonyl-valyl-0-benzyl-tyrosine hydrazide in 300 ml of DMFL, add 53.3 ml (160 mmol) of a 3N solution of hydrogen chloride in ethyl acetate, 4.61 ml (40 ml). mmol) tert.-butyl nitrite in 10 ml DMOL and incubated for 30 minutes at -20 ° C and 10 minutes at -5 ° C. Then the reaction mixture is cooled to -40 ° C and a solution of 16.56 ml ( 120 mmol of triethylamine in 30 ml of DMFL and at -20 C a solution of I isoleucyl-N -benzyl-histidine methyl ester I, obtained by adding 11.04 ml (80 mmol) of triethylamine to 21.7 g (40 mmo) cooled to a solution s) dihydrochloride isoleucyl methyl-N-benzyl-histidine in 200 ml DMFL (.osadok triethylamine hydrochloride was removed by filtration).

The reaction mixture was incubated at + 4 ° C for 24 hours, partially evaporated, the residue was poured into 300 ml of a mixture consisting of a saturated solution of sodium chloride and a 5% solution of sodium bisulfate (2: 1). The precipitate was filtered off, washed with water and dried in vacuum over PjOy / KOH.

The yield of 34.4 g (90%), so pl. 179182 ° С. L -32 (DMFL). , 22 (L); 0.71 (B); 0.81 (B).

2. p-Nitrobenzyl ester of tert.butyloxycarbonyl-phenyl-alash-M -nitro-arginine

Vos-Pye-Lg§ (1GO2) -OB21K02 (W

To cooled to a solution of 7.82 g (20 mmol) of n-nitrobenzyl ester M-nitro-arginine hydrochloride in 50 ml of DMFL, 2,76 ml (20 mmol) of triethylamine, 1.36 g (20 mmol) of imidazole are added in 10 ml of DMFL and 8 g (20 mmol) of tert-butyloxycarbon-1-Fensh-alanine p-nitrophenyl ether in 50 ml of DMFL, then react

The mixture was stirred at room temperature for 48 hours, poured into 5% sodium bisulfate solution and extracted with ethyl acetate.

The ethyl acetate extract was washed with water, 5% sodium bicarbonate solution, water, and evaporated. The residue is triturated with ether, filtered and dried in vacuo over. Yield 10.9 g (91%).

3. Benzyloxycarbonyl-Vgzlil-0-benzyl-tyrosine-1-isoleucyl, -M-benzyl-histidyl-proline Z-Val-Tyr (Bzl) -Ile-His (Bzl) -Pro-NHNH.,. IV. Hydrazide

To a solution of 9.56 g (10 mmol) of benzyloxycarbonyl-valyl-O-benzyl-tyrosyl-isole methyl ester ;; cyl-M-benzyl-histidylproline (P)

5 ml (100 mmol) of hydrazine are added to 200 ml of DMF.

The reaction mixture is incubated at room temperature for 48 hours and poured into 1000 ml of saturated

sodium chloride solution, the precipitate precipitated is filtered, washed with water and dried in a vacuum over

P OS / KOH.

Yield 9.1 g (95%). Rg 0.45 (L); 0.38 (B); 0.70 (D).

4. P-nitrobenzyl ester phenylalanyl-M-nitro-arginine hydrochloride

(V)

HC1-H-Phe-Arg (N0,) - OBzlNO,

To a solution of 10.5 g (19 mmol) of TTT in 100 ml of ethyl acetate, 19 ml (76 mmol) of 4 N, a solution of hydrogen chloride in ethyl acetate, are added. The reaction mixture is kept at 20 ° C for 30 minutes, evaporated in vacuo, the residue is triturated with ether, the precipitate is filtered off and dried in vacuum. above .

Yield 9.9 g (99%). O -6.2 (CH3COOH); I, 0.20 (L); 0.31 (B); 0.48 (D);

5. Benzyloxycarbonyl7-valyl-0-benzyl-tyrosine-1-isoleuc-1-1-benzyl-histidyl-prolyl-phenyl-ananyl-N-nitro-arginine p-nitrobenzyl ester

Z-Val-Tyr (Bzl) -Ile-Hi8 (Bzl) -Pr6-Phe-Lrg (NOg) -OBzlN02 (VI)

2.67 ml (8 mmol) of a 3N solution of hydrogen chloride in ethyl acetate, 0.23 ml (2 mmol) of tert-butyl nitrite are added to a solution of 1.91 g (2 mmol) IV cooled to -20C cooled in -50 ml of DMFL. in 3 ml of DMF and incubated for 30 minutes at -20 ° C and 10 minutes at -5 ° C. Then the reaction mixture is cooled to and the solution is cooled to 1.1 ml (8 mmol of triethylamine in 5 ml of DMF and at -20 ° C. Phenylalanyl-N -nitroarginine p-nitrobenzyl ester obtained by adding 0.28 ml (2 mmol) of triethylamine to a cooled solution of 1.08 g (2 mmol V in 20 ml of DMF. The reaction mixture is kept for 24 hours, poured into a mixture consisting of 150 ml of saturated sodium chloride solution and 50 ml of 5% sodium bisulfate solution.The precipitate formed is filtered off, washed with water and dried in vacuum over 2.5 g (87%). mp. 125127 ° C, -34.0 ° / C 1;: DMF /;, 90 (B); 0.53 (C); 0.68 (D). 6. Valyl-tyrosyl-isoleucyl-histidyl-prolyl-phenylalanyl- arginine Val-Tyr-Ile-His-Pro-Phe-Arg (I) To a solution of 0.5 g (0.35 mmol) VI in a mixture of methanol-acetic acid plant (5: 1: l) was added 0.2 g of palladium black and hydrogenated for 24 hours. Then the catalyst is filtered off, the filter is evaporated, the residue is triturated with ether, the precipitate is filtered watered and purified on a column of cellulose CM-50, eluent-gradient solution of ammonium acetate. After cleaning, the product is lyophilized. Output O ,, 14 g (44%) -24.0 ° (CH, COOH); R, 0.80 (E); 0.77 (F); Amino acid analysis: Val 0.95 (1); Tour 0.90 (1) 1 Not 1.0 (1); His 0.89 (1); Pro 0.97 (1); Phe 0.96 (1); Arg 0.87 (), The biological activity of the described heptapeptide was studied in vitro and in vivo experiments. In vitro experiments studied myotropic activity and in in vivo experiments, the effect on blood pressure of anesthetized rats and the depressor effect of bradykinin. Reanal bradykinin triacetate (WNR) was used as a standard drug. 1. Determination of myotropic activity, Myotropic activity of the described heptapeptide is studied in experiments in vitro by the well-known method by recording isotonic contractions of isolated queens (uterus) of rats. A modified instrument VI6-5MA was used for registration. The agonistic and antagonistic properties of the heptapeptide of formula 1 were studied at a concentration range of -10 M. Parameters characterizing the interaction of the substance with the receptor were determined (l-internal activity, rD — indicator of specific agonistic affinity, pA2 — indicator of specific antagonistic affinity). The time of preliminary exposure of the organs by the studied compound in the study of its effect on myotropism and the effect of bradykinin is 3 minutes. When calculating the concentration-emulation curves, the effect (CCVE) each point or value was determined from 6 experiments ± standard error, 05. The results are presented in the table and in FIG. 1 and 2. Effect of heptapeptide 1 on the myotropic effect of bradykinin; physical pressure. White rats of both sexes weighing 180-200 g are anesthetized with a 25% urethane solution at the rate of 0.5 ml / 100 g of weight. Blood pressure is recorded from the common carotid artery with the help of a transducer (Bentley Trantec Pressure Transducer) on dual-channel pe7

Gemini device (Italy). The substance was injected into the femoral vein as an injection in a volume of 0.1 ml / 200 g at doses of 0.5500 µg / kg. The effect on the depressor effect of bradykinin is studied by administering it in doses of 5-500 µg 1 minute before the introduction of bradykinin.

The toxicity of the described compound is determined in mice.

As a result of the conducted research, it was established that in experiments on vitro on an isolated rat uterus, the heptapeptide of formula 1 exhibits myotropic activity, characterized by the following parameters: ot 0.52.tO, 09, rDg 7.00 + 0.27, and at a concentration of 10M it is competitive inhibits the myotropic effect of bradykinin, 18 ± 0.29, which is almost two times higher than that of the well-known bradykinip antagonist 835608

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu (, 3).

. In in vivo experiments on rats with anesthetized urethane, when administered intravenously, the test compound at doses up to 250 µg / kg does not affect blood pressure and does not affect the depressor effect of bradykinin. At a dose of 500 µg / kg 10, it exhibits an equipressor effect with bradykinin, and also slightly potentiates the depressant effect of the latter,

A LD 5p heptapeptide of formula 1 is 175 ± 15 mg / kg.

Thus, the described compound has a pronounced ability to compete to block bradykinin receptors in extravasal 20 smooth muscle tissues without significantly affecting the hypotensive effect of bradykinin.

(l), 250i KzlH2 BN. {1), 500m / g1 / (GBN

5K

90

70

50

FIG. 2

Claims (2)

Heptapeptide formula ¥ a1-Tug-11e-H13-Pro-Rke-Ag £ possessing the ability to inhibit the myotropic action of bradykinin. 5 and ,, 1083560 one 1083560 2