SU1095587A1 - Cyclic pentapeptide displaying analgetic effect - Google Patents

Abstract

The cyclic Pentapeptide of the formula Tyr-D-Orn-Gly-Phe-Asn has analgesic activity. t SP JV 00

Description

The invention relates to a new biologically active chemical compound, a cyclic pentapeptide, an enkephalin analogue, which has analgesic activity, which can be used in medicine.

Enkefapine, a natural opigate receptor agonist in the brain, was isolated and identified as a mixture of two H1-Ten-peptides: H-Tug-C1u-S1u-Pye-MeOH (methionine-enkephalin) and H-Tyr-Gly Gly-Phe-Leu-OH ( leucine-enkephalin). Both peptides exhibit morphine-like activity in experiments on specific models of the opiate receptor, and also suppress stereospecific binding to the opioid antagonist receptor naloxone in brain homogenates. Met-and Leu-enkephalins show analgesia for 5-15 minutes with intraventricular injection into the brain of the mouse. Cyclic analogs of enkephalin are known, including in the cycle 18, 15 atomic groups (see tab.), However, they show the complete absence of specific enkephalin activity. The closest in chemical structure is the cyclic analog of enkephalin (7) (see table 1), however, the analgesic activity of this compound has not been studied. The aim of the present invention is to expand the arsenal of means of influence on a living organism, the creation of enkephalin analogues with analgesic activity. This goal is achieved by the described cyclic pentapeptide of the formula: Tyr-D-Orn-Gly-Phe-Asn having analgesic activity. Peptide synthesis is carried out using the known methods of peptide chemistry in solution according to the above scheme, using activated tert-butyloxycarbonylamino lot esters, S-amino function of D-ornithine is protected by a benzyloxycarbonyl group, hydroxyl tyrosine - by a tert-butyloxycarbonyl group. The drawing shows a diagram of an Example. For the synthesis of enkephalin analogue 1, amino acids and their derivatives from Reanal (Be gri) are used. All amino acids, except DOrnitine, have L-configuration. The melting temperature was determined in the capillary (without correction). The individual duality of the intermediate compounds is controlled with the help of those on Silufol LVi54 (Czechoslovakia) plates. Glass plates with a fixed layer of SilikaGel 60-F-254 silica gel from Merck (Germany) are used for chromatography of the enkephalin analog. The following solvent systems are used: chloroform - ethanol - acetic acid (AcOH), 85: 10: 5 (A), n - butanol - pyridine - AcOH -, 15:10: 3: 6 (B) i n-butanol - AcOH - N, 0, 4: 1: 1 (C), chloroform - methanol - AcOH 0, 30: 20: 4: 6 (D), 60: 18: 2: 3 (E), n-butanol - pyridine - AcOH - H, 0, 15: 12: 3: 10 (F). The specific optical rotation of the peptides was measured on a Perkin Elmir 141 M polarimeter (HGF). Acid hydrolysis is carried out at 120 ° C for 24 hours. The amino acid composition is determined on a Jeol-3 analyzer; the compounds of elemental analysis are in satisfactory agreement with the calculated C, H, N content. The molecular weight of cyclopeptide is determined by mass spectrometry on a CH-5 Varian MAT (USA) instrument at a temperature of approximately 250 ° C and an ionizing voltage of 70 eV. N-tert-butyloxycarbonyl-Glycyl-phenylalanine (9). 6.6 g (40 mmol) of L-phenylalanine are dissolved in 40 ml of 1N sodium hydroxide solution, 3.34 g of sodium bicarbonate, 50 ml of dimethylformamide (DMF) and 16.94 g (40 mmol) of tert-pentachlorophenyl ether are added. -butyloxycarbonyl-glycine, 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), cooled to 0, and acidified with 0.5 N. hydrochloric acid to pH 3. The ethyl acetate layer is separated, the aqueous phase is extracted again. The combined ethyl acetate layer is washed with water, saturated sodium chloride solution and dried over anhydrous sodium sulfate. The crystalline residue obtained after distilling off the solvent is recrystallized from ethyl acetate with a small addition of petroleum ether. Vkod dipeptide (9) 11.4 g (88%). M.p. 133-35C, And +15.7 (c 1, DMF), Rf 0.73 (A), Rf 0.80 (D). M-tert-butyloxycarbonyl, N ° .Benzyloxycarbonyl-B-ornithyl-glycyl-phenylapanine (11). 10.9 g (33.8 mmol) of the dipeptide (9 is dissolved in 50 ml of a 70% aqueous solution of trifluoroacetic acid (TFA) in dichloromethane and incubated for 30 minutes. The solvent is distilled off, the residue is grown with ether, filtered and washed on the filter and dried in vacuo over potassium hydroxide. 10.4 g (91%) of dipeptvide trifluoroacetate (10) are obtained, Rf 0.63 (D). 5.2 g (15.5 mmol) of trifluoroacetate (10) are dissolved in 50 ml of DMF, cooled to 0 ° C. and cooled to 2. 2, 65 ml (15.5 mmol) of diisopropyl ethylamine in 5 ml of DMF and 8.2 g (15.5 mmol of pentafluorophenyl ether Tg-tert-butane, Loxycarbonyl, N-benzyl sicarbonyl β-D-ornithine dissolved in 15 ml of DMF. The reaction mass is stirred for several hours and, after completion of the reaction, is treated similarly to compound 9. The product obtained after distilling off the solvent is purified on a column of Merck silica gel. Elution is carried out in the system: chloroform- ethanol-ethyl acetate-AcOH-H O, 285: 5: 8: 2: 0.25 The corresponding fractions are collected, evaporated and dried in vacuo. 5.9 g (67%) of protected tripeptide oil (11) are obtained with Rf 0.65 (A), Rf 0.72 (B). N, O-di-tert-butyloxycarbonyl-tyrosyl-N-benzyloxy-carbonyl-D-ornitid-glycyl-phenylalanine (13). 20.0 g (35 mmol) of tripeptide (11) are dissolved in 100 ml of a 50% TFA solution in dichloromethane and incubated for 20 minutes. The solvent is distilled off, the residue is triturated with ether. The precipitate formed is filtered off, washed again with ether and dried in vacuum over K-LIAH hydroxide. 19.2 g (94%) of the tripeptide trifluoroacetate are obtained: (12) cRf 0.62 (D) 18.1 g (31 mmol) of the tripeptide trifluoroacetate (12) are dissolved in 100 ml of DMF, cooled to, added with stirring 5 , 3 ml (31 mmol) of diisopropylethylamine in 10 ml of DMF and 16.6 g (31 mmoles) of N, O-dis-Boc-b-tyrosine pentafluorophenyl ester. The reaction mass is stirred for 1.52 hours (chromatographic control) and after completion of the reaction, the treatments 1 7 are added in the same way as compound 9. The product obtained after distillation of ethyl acetate is stirred with ether and cooled. The precipitate introduced is filtered off, washed with ether and dried in a desiccator. Yield 16.0 (62%) of the protected tetrapeptide (13) with Rf 0.75 (A), Rf 0.55 (B, Merck) + 5.6 (with 1, DMF). K, 0-di-tert-butyloxycarbonylthyroid1- n- (S-benzyloxycarbonyl-asparagyl) -D-ornithyl-glycyl-phenylalanine (15). To 18 g (22 mmol) of the protected tetrapeptide (13) in 150 ml of methanol, 15 ml of AcOH and 20 ml of palladium are added and hydrogenated for several hours. The catalyst is filtered off, the filtrate is evaporated. The residue is triturated with ether and filtered, then re-precipitated from alcohol with ether, washed again with ether and dried in vacuum over potassium hydroxide. 13.8 g (91%) of tetrapeptide (14) are obtained with Rf 0.15 (A) Rf 0.72 (D, Merck) i Rf 0.77 (F, Merck), 5.1 g (6.8 mmol) of tetrapeptide (14) is dissolved in 100 ml of DMF and 1.16 ml (6) are added with stirring and cooling 8 mmol). diisopropylethylamine in 3 ml of DMF and 3.3 g (6.8 mmol) of N -benzyloxycarbonyl-L-asparagine nitrophenyl ester dissolved in 15 ml of DMF. After 2-3 hours, the reaction mass is treated similarly to the compound. 9. The product obtained after distilling off the solvent j is purified on a Merck silica gel column. Elution is carried out in the system: chloroform - ethanol - ethyl acetate - AcOH -, 185: 5: 8: 2: 0.25. The appropriate fractions are collected, evaporated and dried in vacuo. 4.6 g (70%) is obtained with Rf 0.72 (A); Rf 0.83 (D); +8.2 (C 1, DMF). N, 0-di-tert-butyloxycarbonyl tyrosyl-cyclo (-H-B-ornithyl-glycyl-phenylalanyl-asparaginyl) (17), K 700 mg (0.73 mmol) of the protected pentapeptide (15) in 60 ml of methanol, 4 ml of AcOH and 1 ml of water are added to palladium black and hydrogenated for 2-3 hours. The catalyst is filtered off and the filtrate is evaporated. The residue is triturated with ether and dried in vacuo over potassium hydroxide. Receive G 560, mg (94%) of pentapeptide (S) with Rf 0.07 (A), Rf 0.70 (D). 800 mg (0.98 mmol) of pentapeptide (10) are dissolved in 800 ml of purified daFA, cooled to -20 ° C, triethylamine in DMF is added to pH 7.2 and then 0.63 ml (2.94 ml) are added slowly. mmol) diphenylphosphorylazide in 30 ml DMF. The solution is stirred for 7 days and the pH is maintained at about 7 by the periodic addition of triethylamine to the reaction mass. Upon completion of the reaction, the solvent was distilled off at 40 ° C, 1.5-2 ml of ethyl acetate and a small amount of ether were added to the residue. The precipitated precipitate is filtered off, washed with ether, dried in vacuum, and 415 mg of a white powder of a protected cyclic pentapeptide (17) are obtained. Due to poor solubility (17), purification is not carried out.

Chemical shifts of the cyclic pentapeptide H of tyrosyl-cyclo (-N -D-ornithyl-glucyl-phenylalanyl-asyaragink oC). 415 mg (0.52 mmol) of the protected cyclic pentapeptide (17) are dissolved in Go ml of a 70% TIA solution in methylene chloride and incubated for 30 minutes. The solvent is distilled off, the residue is triturated with ether and dried in vacuo. The product obtained is purified by high-performance liquid chromatography on a Du Pont-830 chromatograph. Elution is carried out with a mixture: ethanol-0.1 M ammonium acetate, 13:87. The appropriate fractions are collected and lyophilized. Output: 120 mg (38%) of enkephalin cyclopentapeptide (1), +36 (s 0.5, AcOH) i Rf 0.62 (B, Merck), Rf 0.78 (D, Merck)} amino acid composition: Tug 0 , 93, Gly 1.00; Phe 0.91; Orn 0.95; Asn 0.98.

3.67 4.37 4.61

The magnitude of the calculated molecular weight coincides with its experimental value determined by mass spectrometry (M 595).

The biological activity of the enkephalin analogue (1) was studied in in vivo experiments. Analgesic activity was determined using the H.Ueda method.

In this work, outbred mice weighing 18–22 g were used. The test substance, dissolved in sterile saline, was injected with a U-shaped needle into the cisterna magna of the brain with non-anesthetized micropia in an amount of 10 μl. A dose range of 0.1 to 0.5 µg / animal was tested. Control animals were injected with 10 μl of sterile saline. With

1.4-1.25

2.90

2.56

for intravenous administration, the drug was injected into the tail vein in a range of 15 to 30 mg / kg. The Kazada experimental group consisted of 810 youngsters. The analgesic effect was evaluated by the tail pineh method using an artery clamp applied to the base of the tail. The pain response was determined 5,15,30,60 and 90 min after administration.

Results are expressed by an alternative method as a percentage that showed an analgesic response. When determining ED50 (effective dose of the test substance that causes an analgesic effect in 50% of experimental animals), the method of Litchfield and Wilcoxon is used.

The analgesic activity of the tested compounds is given in Table 2 and 3..

Cyclo- (K ° -B-Ogp, A8p5) -enkephalin. 1 has a pronounced analgesic activity. The maximum effect is observed at the 5th minute after administration. The analgesic activity of the new analogue of enkephalin 10 is significantly JQ, but it exceeds the activity of natural enephalins (2.3) and is 4 times higher than the activity of morphine in molar ratios upon intracisternal administration to mice (see table 2). five

Cyclo- (N ° -D-Orn, Asn) -enkephalin (1), unlike natural enkephalins, is active when administered intravenously. It possesses 60% of the activity of morin when administered intravenously (see 20 tab. 3).

Enkephalin Cycopanalog 1 shows more progenitor analgesic analogs Enkephalin analogue Linear

Cyclo (N-D-Orn 1- With AED 2- The morphine effect was used than the natural enkephalins. Duration of analgesia

A new analogue, (i) is comparable, with the duration of analgesic

effect of morphine with both methods of administration and is 60 minutes (see tab. 2,3)

The effective dose of the described compound (YES) when administered intravenously is 30 µmol / kg, or 22 mg / kg. When administered at a dose of 500 mg / kg, no toxic effects are observed, therefore the therapeutic index of this compound is greater than

500

22 (

 22). which is without 22.

dangerous interval for therapeutic agent.

The results obtained suggest the use of the described compound in medicine.

Table 1 Natural Enkephalins. Analgesic activity of cyclo- (K-D-Orn, Asn) -enkephalin upon intracysterial administration to mice (tail pineh method) Table 2 Hydrochloride produced in Tashkent HFZ.

Analgesic activity of cyclo- (L-D-Orn, Asri) -Enkephalin when administered intravenously

Cyclo- (H -D-Orn Asn) Enkephalin30 (18-51)

Morphine 18 (15-22)

Table3

60

60 60

5 5-15 100

Claims (1)

Cyclic Pentapeptide Formula Tug-P-rgp-O1u-Pl-Az-possessing analgesic activity. 1095587 A1