SU1341970A1 - Analog of enkephalin displaying analgetic activity - Google Patents

Abstract

The invention relates to peptide derivatives, in particular an analogue of znkefalin of the formula Tyr-D-grn Gly Phe. The invention relates to an Enkephalin I analogue Tug-D Ogpe-Gly-Phe-NH Arg-Asn-1, a new biologically active compound with analgesic activity that can find application in medicine. The purpose of the invention is to search for a number of analogue-analog peptides with low toxicity and higher analgesic activity. The enkephalin analogue described (smoshenu in the drawing) is synthesized by known methods of peptide chemistry in solution, according to the scheme presented, using activated tert-butyloxycarbonylamino NHH esters, which has biological activity and can be used in medicine. The goal is to create more active substances of the specified class. The preparation is carried out using activated tert-butyloxycarbonylaminoacid esters with amino protection. The tests show that the new analogue of enkephalin is low toxic with internal administration 1.5 times more active than morphine, 3 times more active than analog: () -, and with intracisternal administration to mice, 3 times more active than morphine . 1 il. 2 tab. acids. J-Amino function B of ornithine is protected by a benzyloxycarbonyl group, hydroxyl tyrosine of a t-butyloxycarbonyl group. It was established that amidation of the carboxyl group in the hexapeptide results in an unexpected sharp increase in the analgesic activity of the obtained enkephalin analogue, both in intracnsternal and intravenous administration in animal experiments. For the synthesis of the enkephalin analogue (I), amino acids and their derivatives from Reanal (Vengry ). All amino acids, except for D-ornithine, have the L-configuration. The melting point of the substances is determined in the capillary (without correction). The personality of the intermediate connects is controlled with the help of those on plates M w,

Description

kah Silufol (Czechoslovakia). For chromatography of an enkephalin analogue, glass plates with a fixed layer of Silica Gel 60 F-254 Silica Gel from Merck (Germany) are used in the following systems: chloroform – ethanol – acetic acid (AcOH), 85; 10g5 (A); n-butanol-pyridine-AcOH-H 0 30 g 20: 4: 6 (D); The specific optical rotation of the peptides is measured on a Perkin-Elmer 141M polarimeter (HGF). Acid hydrolysis is carried out at 20 s for 24 hours. The amino acid composition is determined on an analyzer. For all compounds, the elemental analysis data agree well with the calculated C, AND, N content.

Example K-tert-Butyloxyrsarbonyl-glycyl-phenylalanine (7).

6j6 g (40 mmol) of L-feschalanine is dissolved in 40 ml of J n, sodium hydroxide solution, g of sodium bicarbonate, 50 ml of dimethylformamide (DMF) and 16.94 i (40 mmol) of tert-butyl oxycarbonyl ether are added. -glycine5 dissolved in 20 ml of DMF. The mixture is stirred for several hours. After completion of the reaction (chromatographic control) —the reaction mass is diluted with ethyl acetate and water (before separation of the layers) 5 is cooled to 0 ° C and acidified with 0.5 H hydrochloric acid to pH 3, the ethyl acetate layer is separated, the aqueous phase extract it again with combined ethyl acetate and water with saturated sodium chloride solution and dry over anhydrous sodium sulfate. The crystalline precipitate obtained after distilling off the solvent recrystallized from ethyl acetate with a small addition of petroleum zfirao. Dipeptide yield (7) 11.4 g (88%) 5 mp. 133-135 ° C; , 7 ° (с „1, DMF), R 0.73 (A), 0.80 (D). ,

N-tert-butyloxycarbonyl Ld N - benzyloxycarbon1 l B-ornitol | Shglycyl feschalanine (9), 10.9 g (mmol) of the dipeptide (7) are dissolved in 50 ml of a 70% solution of trifluoroxy acid (TFA) in dichloromethane and quenched for 30 minutes. The solvent is distilled off, the residue is triturated with ether, filtered off T; washed with ether on a filter and dried in vacuo over potassium hydroxide; 10.4 g () of dipeptide trifluoroacetate (8), Rj, are obtained. (D)

5.2 g (15, p5 mmol) of trifluoroacetate

(8) is dissolved in 50 ml of DMF, cooled before and "while cooling is added

2.65 ml (1555 mmol) of diisopropyl-amine in 5 ml of DMF and 8.2 g (15.5 mmol) of M-t-butyloxycarbonyl pentafluorophenyl ether, N-benzyloxy-9-bonyl-B-ornithine J dissolved in

15 MP daFA. The reaction mixture is stirred for several hours and, after completion of the reaction, is treated similarly to compound 7. The product obtained after distilling off the solvent is purified on a Merck Silgel column. Elution is carried out in a chloroform system. - ethanol - ethyl acetate-AcOH-H O 285: 5: 8: 2: 0.25, The corresponding fractions are collected, evaporated and the distillate is in a vacuum. Get g (67%) oil protected tripeptide

(9) with R 0.65 (A), 0.72 (B).

N.0-di-tert-butyloxycarbonyl-rosil-N-benzyloxy-sarbonyl-B-orni-,

Tyl-glycyl-phenylalanine (11),

20.0 g (35 mmol) of tripeptide (9) is dissolved in 100 ml of a 50% TFA solution in dichloromethane and held for 20 minutes. The solvent is distilled off the residue triturated with ether. The precipitate formed is filtered off, washed again with ether and sucked in tzakuma over potassium hydroxide. 19.2 g (94%) of trifluoroacetate are obtained.

Tripeptide (10) J R. 0.62 (D).

IBjl g (31 mmol) of tripeptide trifluoroacetate (0) is dissolved in 100 ml, the CMF is cooled to 0 ° C, 5 |, 3 ml (31 mmol) are added with stirring

diisopropylethylamine in 10 ml of DMF and 16., 6 g (31 mmol) of N, 0-di-Boc-L-tyrosine pentafluorophenyl ester. The reaction mass is stirred 1, (chromatographic control) and after completion of the reaction is treated similarly to compound 7. The product j obtained after distillation of ethyl acetate j. stir with ether and cool. The precipitate formed is filtered off, washed with ether and dried in a desiccator. The yield is 16.0 g (63%) of the overclocked tetrapeptide (P); R 0.75 (A); (AT,

 Merck); From -b (p. 1, .CMFA),

H, .0-di t ret-butyloxycarbonyl-tyrosyl-N (1h benzshüksicarbonasp raginsh1) -B-ornithyl-glytssh1-phenylalanip (13) s, K 18 g (22 mmol) of the protected tetrapeptide (I) in 150 ml

methanol, 15 ml of AcOH and 20 ml of H, 0 to add palladium black and hydropyTOT within a few hours. K - the analyzer is filtered, the filtrate is evaporated. The residue is triturated with ether and the filter is sweat, then the precipitate is re-precipitated, extracted from alcohol with ether, washed again with ether and dried under vacuum over potassium hydroxide. Obtain 3.8 g (91%) of tetrapeptide 12) j R, 0.15 (A) 0.72 (D, Merck); 0.77 (C, Merck),

5.1 g (6.8 mol) of tetrapeptic acid (12) is dissolved in 100 ml of DMF and 1.16 ml (6.8 mmol) of diisopropylzyl amine in 3 ml of DMF and 3.3 g (6 ml) are added with stirring and cooling. , 8 mmol) of nitrophenyl ether M benz1-shoxycarbonyl-b-asparagia, dissolved in 15 ml of DMF. After 2-3 hours, the reaction mass is treated similarly to compound 7. The product obtained after distilling off the solvent is cleaned on a silica gel column of Merck , Elution of the conductor with 5 tons. In the system chloroform - ethanol - ethyl acetate - AcOH-HjQ 185: 5: 8: 2: 0.25. The appropriate fractions are collected, evaporated and dried in vacuo. Obtain 4.6 g (70%) of the branched entaphalin pentapeptide (13) with M + 8.2 °, (p. 1,. 0.72 (A) j 0.83 (D),

N, 0-di-tert-butyloxycarbonyl-tyrosyl- N - (tribenzyloxycarbonyl-argnyl-asparaginyl) -D-ornithyl-glycyl-phenylalanine (15), From 2 g i (2.28 mmol) of pentapeptide (13) and 1, 69 g (2.28 mol) of tribenyloxycarbonyl-b-arginine pentafluorophenyl ester give hexapeptide (15) similar to compound (13). Purification of the product obtained after distilling off the solvent does not lead to poor solubility of the obtained compound. The yield of 2.7 g of protected hexapeptide (15),

K, 0-di-tert-butyloxycarbonyl-1-thyrosyl-H - (tribenzyl-hydroxycarbonyl-arginyl-asparaginyl) -D-ornithyl-glycylphenylalaninamide (16) To a solution of 0.514 g (0.373 mmol), compounds (15 ) 0.050 g (0.373 mmol) of 1-hydroxybenzotriazole ammonium salt and 0.076 g (0.373 g-shol) are added to 10 ml of DMF

0.352 g (68%) of Zy1chidenny amrod. peptide hex (16); R, 0.75 (A). Due to the poor solubility, the chromatographic purification of the compound (16) is not carried out. js

Tyrosyl-N - (arginyl-asparticyl-D-ornithyl-glcylphenylalaninamide (1), To a solution of 0.352 g (0.256 mmol) protected 10 hexapeptide (16) in 15 ml of methanol, 1.5 ml of AcOH and-0.5 nl of water added pelladium blacks are hydrogenated and hydrated for 2-3 hours, the catalyst is filtered off, the filtrate is evaporated.The residue is triturated with ether and dried in vacuo over potassium hydroxide, Paul, the dissolved product is dissolved in 5 ml of a 70% TFA solution in methylene chloride and incubated for 30 minutes

20 The solvent is distilled off and the residue is triturated with sir. The precipitate formed is filtered off, washed with effum and cyiuaT in vacuo. The resulting product is purified by highly efficient

25 by liquid chromatography on a Lorbax Cg column. Elution is carried out with an ethanol-O mixture, a 1 M solution of ammonium acetate 5:95. The appropriate fractions are collected and lyophilized. A course of 0.076 g (38%) of branched amy hexapeptide (1). P +45 (s; 0.2 n-AcOH); R 0.43 (B, Merck 0521 (D, Merck); amino acid composition: Tyr 0.93; Gly 1.00; Phe 1.07; Orn 0.97; Asn 1.03; Arg 1.00,

The biological activity of the enkephalin described was studied in in vivo experiments using the Uzda method on outbred male mice weighing 18-22 g. The analgesic effect was studied in comparison with that of morphine, natural enkephalins (Leu and Met enkephalin), a structural analogue of the enkephalin described.

When administered intracisternally, the test compounds dissolved in sterile saline in a volume of 10 µl were injected with a J-shaped needle into a cisterna manga brain 50 hectares to unanesthetized mice. The dose range from 0.0005 to 0.01 µg / animal was tested. t 10 µl of sterile physiological, logical solution. With intravenous35

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dicyclohexylcarbodiimide (DCGC), by the administration of Fe-55 P, the drug is introduced into the tailing mixture overnight. After completion of the reaction, the reaction mass is filtered off and treated in the same way as compound 7

3419706

0.352 g (68%) of Zy1chidenny amrod. hexa-peptide (16); R, 0.75 (A). Due to the poor solubility, the chromatographic purification of the compound (16) is not carried out. js

Tyrosyl-N - (arginyl-asparticyl-D-ornithyl-glcylphenylalaninamide (1), To a solution of 0.352 g (0.256 mmol) protected 10 hexapeptide (16) in 15 ml of methanol, 1.5 ml of AcOH and-0.5 nl of water added pelladium blacks are hydrogenated and hydrated for 2-3 hours, the catalyst is filtered off, the filtrate is evaporated.The residue is triturated with ether and dried in vacuo over potassium hydroxide, Paul, the dissolved product is dissolved in 5 ml of a 70% TFA solution in methylene chloride and incubated for 30 minutes

20 The solvent is distilled off and the residue is triturated with sir. The precipitate formed is filtered off and washed with ether and cyiuaT in vacuo. The resulting product is purified by highly efficient

25 by liquid chromatography on a Lorbax Cg column. The elution is carried out with an ethanol-O mixture, a 1 M solution of ammonium acetate 5:95. The appropriate fractions are collected and lyophilized. A course of 0.076 g (38%) of branched amide hexa-peptide (1). P +45 ( s; 0,2 n-AcOH); R 0.43 (B, Merck) 0521 (D, Merck); amino acid composition: Tug 0.93; Gly 1.00; Phe 1.07; Orn 0.97; Asn 1.03; Arg 1.00,

The biological activity of the enkephalin described was studied in in vivo experiments using the Uzda method on outbred male mice weighing 18-22 g. The analgesic effect was studied in comparison with that of morphine, natural enkephalins (Leu and Met enkephalin), a structural analogue of the enkephalin described.

When administered intracisternally, the test compounds dissolved in sterile saline in a volume of 10 µl were injected with a J-shaped needle into a cisterna manga brain 50 hectares to unanesthetized mice. The dose range from 0.0005 to 0.01 µg / animal was tested. t 10 µl of sterile physiological, logical solution. With intravenous35

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45

Tov vein mice in the dose range from 5 to 20 mg / kg animal weight. Each experiment: entalna, the group consists of 10 mice. Analgesic effect

Compounds are evaluated for tail pinch dough using an arterial clamp that is applied to the base of the tail. The pain response was determined 5, 15, 30, 60, 90 minutes after administration and then every 30 minutes until the analgesic reaction disappeared.

The results are expressed by an alternative method in the percentage of mice that showed an analgesic reaction. In calculating EDo, the method of Litchfield and Wilcoxon, Analgesic activity and duration of action are used.

compounds are given in table, 1 and 2,

one

The new analogue of enkephalin exhibits pronounced analgesic activity. The maximum effect is observed at 5 minutes after administration. As can be seen from tabLO, the analgesic activity of the enkephalin analog (1) when administered intracisternally to mice significantly exceeds the analgesic activity of natural enkephalins (compound 3, 4) and 12.5 times the activity of compound 2 (analog), the described compound 1 is 300 times more active than morphine (compound 6), 25 times more active than the natural long opioid peptide-B-endorphin (compound 5 and 3 times more active than the well-known potent enkephalin analogue FK-33 824 (compound 7),

Analgesic activity of enkephalin analogs when injected intracisternally into mice (tail pinch method)

Tyr D-Orn Gly-Phe NH

Arg-Asn-J Tyr-D-Orn-Gly-Phe

Arg-asn-j

(analog-comparison); Leu-Enkephalin iMet-Enkefapine B-endorphin

Unlike natural enkephalins, the described compound is actively administered intravenously (table 2). The analgesic activity of a new branched, hexapeptidamine 1 is 150% of the morpholine activity when administered intravenously, and it is 3 times more active than its analog (compound 2) with this method of administration .

The therapeutic index of the novel compound (1) is B0Q / 200. more than 18 vGG) because doses up to

200 mg / kg (compound 1) do not yet cause any toxic effects or death after intravenous administration.

Thus, the described compound 1 has a high analgesic activity, is superior to natural analgesics and well-known analogs in structure and, at the same time, has a low toxicity,

Claims (1)

Invention Formula Ankefalin analogue of the formula Arg-Asn has analgesic activity, Table 1 0.004 (0, 005) 300 0.05 (0.03-0.10) 24 174 (102-281) 134 (90-261) 0.096 (0.058-0.157) 33-824) 5-151.2 (0.6-2.2) 0.013 Note, an effective dose that causes an analgesic reaction in 50% of animal life. Morphine hydrochloride was used in ampoules produced by the Tashkent HFZ. table 2 Analgesic activity of enkephalin analogue when administered intravenously to mice Arg-Asti J Morphine 18 (15-22) 5-15 Note When ED 1 92 60 100 V Editor L.Narodna Tehred M.Hodanich Order 997 Circulation 319 Subscription VNIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Raushsk nab, De 4/5 Production and publishing combine Patent, g, Uzhgorod, ul, Gagarin, 101 etf W Proofreader Oh, Kravtsov