CZ7038U1 - Dalargin analogues having N-methylated peptide bond - Google Patents

Description

Dalargin analogs with N-methylated peptide bond

Technical field

The invention relates to Dalargin analogs having an N-methylated peptide bond between amino acids at positions 3 and 4.

BACKGROUND OF THE INVENTION

The brain pentapeptide enkephalin was isolated in 1975 (Hughes I. et.al., Nature 258, 577, 1975). In order to stabilize the peptide structure, analogs have been prepared with a number of variations preventing, for example, by making peptide bonds unavailable by enzymatic cleavage by insertion of sterically stressed amino acids (Pospíšek J. et al., AO 270617). Dalargin and its stabilized analogs have also been of practical interest, for example, in the rearing of fish fry as anti-stress agents (Kouřil J. et al., AO 272150 and U.S. Patent No. 276,916).

AND

Tyr-Gly-Gly-Phe-Leu

The enzyme enkephalinase (dipeptidylcarboxypeptidase) appears to be responsible for cleaving the same bond in Dalargin.

Tyr-D-Ala-Gly-Phe-Leu-Arg after loss of carboxyterminal arginine by carboxypeptidase

B.

The blocking of the cleavage of this bond in Dalargin can be accomplished by substituting the glycine at the 3-position with D-alanine and the like.

> L-phenylalanine, which should be a sufficient measure in the stereospecificity of enzyme activity. However, these synthetic substitutions are "reflected differently at the level of the respective receptors.

The essence of the technical solution

The present invention relates to Dalargin analogues having an N-methylated peptide bond between amino acids at positions 3 and 4 of the peptide of formula I

L-Tyr-D-Ala-Gly-XL-Leu-L-ArgNH ₂ (I) wherein X is N * -methyl-L-phenylalanine or

N, N-methyl-D-phenylalanine.

The analogs of formula (I) are prepared by synthesizing the peptides by sequentially chain building on a p-methylbenzhydroamine resin from Boc-amino acids. The final product is cleaved from the resin with trifluoroacetic acid. The crude product was purified by RP HPLC. Substances released from HF resin and purified by HPLC have a discriminated binding stabilized against enzyme systems.

-1CZ 7038 Ul

Examples

Example 1

Preparation

Boc-N-methyl-D-phenylalanine

To a mixture of 50.4 mL of tetrahydrofuran and 5.6 mL of dimethylformamide was added 5.3 g of Boc-D-phenylalanine. 2.88 g of an oil suspension of sodium hydride were added with stirring and after 10 minutes 10 ml of methyl iodide. The entire reaction mixture was heated to 80 ° C for 24 hours. Hydrolysis was then carried out and the product was taken up in diethyl ether. The ether solution was dried over anhydrous magnesium sulfate, and after filtering off the desiccant, the ether was distilled off in vacuo. To the residue were added 20 mL dioxane and 20 mL 2M NaOH. The reaction mixture was stirred at room temperature for 2 hours, then acidified with citric acid and the product was extracted into ethyl acetate. The ethyl acetate product was dried over anhydrous magnesium sulfate, and after filtering off the desiccant, the solvent was distilled off. The residual oil was dissolved in ether and the product precipitated as a salt with dicyclohexylamine.

Yield 5.84 g, i.e. 63.47%. BT 154-155 ° C. For C ₂₇ H ₄₄ N ₂ O ₄ (461.4) calculated C 70.40, H 9.63, N 6.08, found C 70.58, H 9.57, 5.35%. N.

The mass spectrum shows M ⁺ 461.

·>

and

Example 2

Preparation of Boc-N-methyl-L-phenylalanine

To a mixture of 75.6 mL of tetrahydrofuran and 8.4 mL of dimethylformamide was added 8.0 g of Boc-D-phenylalanine. 4.32 g of an oil suspension of sodium hydride were added with stirring and after 10 minutes 15 ml of methyl iodide was added. The entire reaction mixture was heated at 80 ° C for 24 hours. Hydrolysis was then carried out and the product was taken up in diethyl ether. The ether solution was dried over anhydrous magnesium sulfate, and after filtering off the desiccant, the ether was distilled off in vacuo. To the residue was added 30 mL dioxane and 30 mL 2M NaOH. The reaction mixture was stirred at room temperature for 2 hours, then acidified with citric acid and the product was extracted into ethyl acetate. The ethyl acetate product was dried over anhydrous magnesium sulfate, and after filtering off the desiccant, the solvent was distilled off. The residual oil was dissolved in ether and the product precipitated as a salt with dicyclohexylamine.

Yield 8.94 g, i.e. 70.0

H, 9.63; N, 6.08. 5.34%.

For ^C 27 ^H 44 ^N 2 O4 ^{(461, 4)} calculated N Found 70.52% C, 9.52% H,

Example 3

Preparation of L-Me (Phe ⁴ ) -Dalargine

-2CZ 7038 Ul

Protected on p-methylbenzhydroamine resin was prepared by sequential synthesis

L-Tyr-D-Ala-Gly-L (Me) Phe-Leu-Arg NH ₂

The amino acids of the individual amino acids were protected by the Boc group and the quanidine group of arginine by the tosyl group and the hydroxyl group of the tyrosine by the 2,6-dichlorobenzyl group. A three-fold excess of individual amino acids was used in the synthesis. From the resin, the peptide and the protecting groups were cleaved with anhydrous HF in the presence of anisole. After HF was distilled off, the reaction mixture was stirred in 50% acetic acid, the resin was filtered off, washed with water and the combined filtrates were lyophilized. The peptide mixture was purified by HPLC.

Example 4

Preparation of D-Me (Phe ⁴ ) -Dalargin

Protected on p-methylbenzhydroamine resin was prepared by sequential synthesis

L-Tyr-D-Ala-Gly-D (Me) Phe-Leu-Arg NH ₂

The amino groups of the individual amino acids were protected by the Boc group and the quanidine group by arginine by the tosyl group and the hydroxyl group by tyrosine by the 2,6-dichlorobenzyl group. A three-fold excess of individual amino acids was used in the synthesis. From the resin, the peptide and the protecting groups were cleaved with anhydrous HF in the presence of anisole. After HF was distilled off, the reaction mixture was stirred in 50% acetic acid, the resin was filtered off, washed with water and the combined filtrates were lyophilized. The peptide mixture was purified by HPLC.

Claims (1)

PROTECTION REQUIREMENTS Dalargin analogs with N-methylated peptide bond between amino acids at positions 3 and 4 of peptides of formula I L-Tyr-D-Ala-Gly-XL-Leu-L-ArgNH ₂ (I) wherein X is -methyl-L-phenylalanine or -methyl-D-phenylalanine.