CS270617B1 - Enkephalin's analogues and method of their preparation - Google Patents

Abstract

The subject of the invention are novel enkephalin analogs - derived from Dalargin, with non-code an amino acid of sterically demanding nature; side chain of formula I L-Tyr-D-Ala-Gly-L-Phe-X-L-Arg (I), where X = L-neopentylglycine (L-Neo) or X = D-neopentylglycine (D-Neo) and a method for preparing the same. It rests wherein the terminal protected tripreptide is condensed with a C-terminal tripeptide containing D, L unnatural amino acid. Hexapeptide is separated after deprotection high performance chromatography on its diastereoisomers. These substances have higher opioid levels effects than Dalargin and derivative with D-Neo in position 5 has a protracted effect.

Description

The invention relates to synthetic analogues of enkephalin, an opioid pentapeptide first isolated and characterized by Huges in 1975 (Huges J. et al., Nature 258, 577, 1975).

Thousands of enkephalin analogues have been prepared to date. Many syntheses have been motivated by the preparation of analogs with increased affinity for different receptor populations, increased selectivity of action, and greater metabolic stability.

One approach to synthesis is the replacement of natural amino acids with sterically demanding amino acids, whether O- or L-forms, which results in a reduction in the number of conferences of the peptide being prepared.

As part of this research, enkephalin analogs modified at position 5 were also prepared. L-tert-leucine, which has a bulk target, a butyl side chain, was introduced into position 5 (Pospíšek J. et al., Collection Czechoslov. Chem. Commun. 52 , 1867, 1987).

The use of this non-coding amino acid resulted in greater stability to enzymatic cleavage and 2-3 times higher activity in monitoring opiate activity than D-Ala, Leu-enkephalin (Fauchere JL, Petermann C., Chim. Acta 63, 824; 1980).

The work was focused on the change of position 5 in the known active analogue enkephalin Dalargin, L-Tyr-D-Ala-Gly-L-Phe-L-Leu-L-Arg (Titov ML et al., Bull. Vsesoyuz. Cardiolog. Nauch. Central AMN USSR 2, 72, 1985). In this work, the synthesis of Dalargin analog, (L-Neo) Dalargin and (D-Neo) Dalargin is presented, indicating the structure in which L-leucine was replaced by L- or D-neopentylglycine.

The present invention relates to novel Dalargin-derived enkephalin analogs with a non-coding amino acid with a spherically demanding side chain of formula I L-Tyr-D-Ala-Gly-L-Phe-XL-Arg, (I) wherein X = D-neopentylglycine or L-neopentylglycine , and a process for the preparation of enkephalin analogs derived from Dalargin of general formula 1, characterized in that the N-terminal tripeptide L-tyrosyl-D-alanyl-glycine protected by a tert-butyloxycarbonyl group is condensed with the C-terminal tripeptide L-phenylalanyl-D, L- neopentylglycyl-L-arginine and the protected hexapeptide are separated into their diastereoisomers by high performance liquid chromatography after cleavage of the protecting group with trifluoroacetic acid.

Substances prepared by fragment condensation of tripeptides were first purified by silica gel column chromatography. After deprotection with TFA, the resulting product was separated by preparative chromatography on C-18 into its diastereoisomers. Both substances are □ pioidly more potent than Oalargin, with the D-form showing a prolonged effect.

Example 1 5 5 (L-Neo) -Dalargin and (D-Neo) -Dalargin

To a stirred solution of 0.72 g (1.13 mmol) of (o-tert-butyloxycarbonyl) -L-tyrosyl-D-alanyl-glycine p-nitrophenyl ester in 10 mL of dimethylformamide was added 0.48 g (1.13 mmol) of L-phenylalanyl -D, L-neopentylglycyl-L-arginine. The reaction mixture was stirred at room temperature for 24 hours. Then, dimethylformamide was evaporated in vacuo, and the residue was dissolved in chloroform and chromatographed on a silica gel column with chloroform-methanol (7: 3).

CS 270617 Bl

The main fraction was evaporated to dryness in vacuo and the solid was recrystallized from ethanol-ether; 0.86 g of substance was obtained, i.e. 73%, m.p. 180-182 ° C. Amino acid analysis: Tyr: 1.06; Ala: 1.07; Gly: 1.06; Phe: 1.01; Neo: 1.00; Arg: 0.98.

The tert-butylosycarbonyl group was cleaved off in the usual manner with trifluoroacetic acid to give 0.6 g after isolation; i.e. 96% of the substance which was separated by preparative chromatography (HPLC) on a C-18 silasorb column using a gradient (0-100%) of methanol-water into two diastereoisomeric substances: (L-Neo ³ ) -alargin and (D-Neo ³ ) -0alargin. '.

Amino acid analyzes: (L-Neo-Dalargin: Tyr: 1.00; Ala: 1.10; Gly: 1.19;

Phe: 0.99; Neo: 1.00; Arg: 0.92.

(D-Neo ⁵ ) -alargin: Tyr: 1.01; Ala: 1.02; Gly: 1.02;

Phe: 1.01; Neo: 1.00; Arg: 0.98 ..

Example 2.

Male guinea pigs weighing 200-300 g were used to test Dalargin derivatives. About 8 cm of ileum was operated on at a distance of about 7 cm from the appendix from animals stunned and killed by ligament rupture. The upper mucosal layer was carefully separated from the intestine and fixed in a test vessel with a pneumoxide-aerated nutrient solution containing in 1 liter: 120 mmol / l NaCl, 5.9 mmol / l KCl, 15.4 mmol / l NaHCO 3, 1.2 mmol / l NaH ₂ PO ₄ , 2.5 mmol / l CaCl ₂ , 1.2 mmol / l MgCl 2? and 2-g glucose. The medium temperature was maintained at 33 ° C. The mucosal layer of the ileum was surgically sutured to a magnetoelectric contraction sensor that was connected to a recording device. After 20 minutes, a voltage of 1 pulse / 10 s and an amplitude of 8 V / l cm of distance between the electrodes was applied to the platinum electrodes in the vessel.

Dalargin was used as a standard for testing (L-neopentylglycine ³ ) -Dalargin and (D-neopentylglycine ^{3) -Dalargin.} From a 1 mg / ml solution, dilution series of 10 to 10 ^-3 mg / ml were prepared by diluting with nutrient medium. The reduction in ileum contractions was measured over a dose range such that at least one dose corresponded to a greater and one less than 50% inhibition. After each dose, the vial was rinsed 3 times with nutrient solution and the next dose was administered only after the contractions had stabilized at the original level before the dose. From the plot of the magnitude of the contractions on the log dose (mg), the dose corresponding to 50% inhibition was found, which was converted to the appropriate concentration of the substance, referred to as IC 50 (mol / l). The following Table I summarizes the IC ₅₀ found for Dalargin and its neopentylglycine derivatives from 6 independent measurements.

Tabu lkal

Values 10 g-g for Dalargin, (L-Neo ³ ) -Oalargin and (D-Neo ³ ) -Dalargin

Substance IC 50

Dalargin · '380 í 30 (L-Neo ³ ) -0alargin' 20 i 7 (D-Neob-0alargin 85 - 10

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The results show about 4-fold higher biological activity of (D-Neo ⁵ ) -Dalargin and 20-fold higher activity of L-Neo-β-Dalargin compared to Dalargin. At the same dose of 2 x 10 ' mg, the fading time of the D-form was twice that of the L-form.

Claims (2)

OBJECT OF THE INVENTION Dalargin-derived enkephalin analogs with a non-coding amino acid with a sterically demanding side chain of formula I, L-Tyr-D-Al-α-Gly-L-Phe-X-L-Arg, (I) where X = D-neopentylglycine or L-neopentylglycine 2. A process for the preparation of analogues of general formula I, according to * 1, characterized in that the N-terminal tripeptide L-tyrosyl-D-alanyl-glycine · protected by a tert-butyloxycarbonyl group is condensed with the C-terminal tripeptide L-phenylalanyl-D, L -neopentylglycyl-L-arginine and the protected hexapeptide are separated into their diastereoisomers by high performance liquid chromatography after cleavage of the protecting group with trifluoroacetic acid.