CZ262796A3 - Cyclosylated n-protected analogs of dalargin and process for preparing thereof - Google Patents

Abstract

X-Tyr (OY) -D-Ala-Gly-Phe-Leu-ArgOHI 2), where X is phenylacetyl or a target. butyloxycarbonyl; Y is hydrogen when Z is 4- (Obeta-D-galactopyranosyl-D-deoxy-1) -N-amine oglucitol (substances I, II) or thiogalactosyl, T if Z is hydrogen (substances III, IV). General Fabrics The CO of formula I is prepared so that it is N-protected dalargin analogs are treated with reducing sugar in the presence of sodium cyanoborohydride (substances I and II) or chloroethylthiogalactopyranoside (substances III and IV).

Description

(57)

X-Tyr (OY) -D-Ala-Gly-Phe-Leu-ArgOHIZ) where X is phenylacetyl or tert. butyloxycarbonyl; Y is hydrogen when Z is 4- (Obeta-D-galactopyranosyl-D-deoxy-1-N-aminoglucitol (compounds I, II) or thiogalactosyl,

T if Z is hydrogen (compounds III, IV). The compounds of formula I are prepared by treating the N-protected analogues of dalargine with a reducing sugar in the presence of sodium cyanoborohydride (compounds I and II) or chloroethyl thio galactopyranoside (compounds III and IV).

CZ 2627-96

Glycosylated N-protected analogues for the preparation of dalargin and a method thereof

Technical field

The invention relates to glycosylated N-protected analogues of dalargin and a process for their preparation.

BACKGROUND OF THE INVENTION

Dalargin functional groups such as hydroxyl tyrosine, its terminal amino group, and the quanidyl group of arginine are targets of glycosylation modifications. The possibilities of modification of dalargin by sugar residues are far from exhausted, not only due to the presence of carboxyl (Š.Horvát et. Al. Helv.Chim.Acta 74, 951-955, 1991) and its modification by glycosides or by insertion of amino acids with another modifiable functional group into original peptide structures (R.Polt et al. J.Amer, Chem. Soc. 114, 10249-1992). Using reductive amination, an approach aimed at modifying the NH ₂ or NH group in the guanidyl group of arginine does not reduce the number of possible products to (compared to glycosylation carried out in dalargine with haloalkylthioglycoside), two monoglycosylated and one diglycosylated, and even product separation especially from a preparatory point of view. In an effort to clarify the reaction, the authors prepared two N-protected analogues of dalargin, Boc-dalargin and Pac-dalargin. Boc (Benzyloxycarbonyl) and Pac (Phenylacetyl) dalargin derivatives effectively block the primary amino group of tyrosine and are removable under various conditions, with Boc trifluoric acid and enzymatically catalysed hydrolysis (penicillin amidohydrolase) Pac group

of tyrosine. This approach gives the possibility to prepare dalargin derivatives glycosylated at the guanidyl group of arginine at position 6.

The essence of the invention

The present invention provides glycosylated N-protected dalargine analogs of Formula I

OY z

X-Tyr-D-Ala-Gly-Phe-Leu-ArgOH (I) wherein X is phenylacetyl or a target. butyloxycarbonyl, *

Y is hydrogen when Z is

4- (O-beta-D-galactopyranosyl-D-deoxy-1-N-aminoglucitol (I, II), or thiogalactosyl when Z is hydrogen (III, IV).

Compounds of formula I are prepared by treating N-protected analogues of dalargine with a reducing sugar in the presence of sodium cyanoborohydride (compounds I and II) or chloroethylthiogalactopyranoside (compounds III and IV).

Preferably, compounds I and II are prepared by treating a Pac dalargin, a precursor of i, or Boc dalargin, a precursor of II, dissolved in a concentration of 1-20 nM (in 50% EtOH) in a neutral pH buffer with a reducing sugar (10-50 mg / ml) in the presence of scidium cyanoborohydride (10 mg / ml) at 37 ° C for 24 hours. Upon completion of glycosylation, the reaction is stopped by acidification, for example with 50% acetic acid. Compounds III and IV are prepared by treating the precursors of phenylacetyl or tert-butyloxycarbonyl dalargine with chloroethylthiogalactopyranoside. The isolation of the reaction products is preferably carried out by separation on a molecular network.

IN

DETAILED DESCRIPTION OF THE INVENTION

Example 1.

N-Phenylacetyl dalargin (5mg) was dissolved in 1 ml of physiological saline containing 50% EtOH 0.05 M Na-phosphate, pH 7.5. 30 mg of lactose and 10 mg of sodium cyanoborohydride were added to the solution. The mixture was left at 37 ° C for 24 hours and then quenched by acidification with 50% acetic acid. The reaction mixture was separated on a Biogel P-2 column (1.5 x 45cm) that was equilibrated with 0.05M NH ₄ HCO ₃ . The column was eluted with the same ammonium bicarbonate solution at a rate of 8 ml / h, fractions were collected by 2 ml. During the elution, the presence of peptides (280 nm) and free sugar was monitored (Dubois et al. Anal.Chem. 18, 350, 1956). Glycosylated derivatives of dalargin were separated by HPLC and their presence monitored by mass spectroscopy, capillary electrophoresis and biol. GPI test.

Example 2.

N-tert-butyloxycarbonyl dalargine (5 mg) was treated as described in Example 1. Separation and analytical methods were identical to Example 1.

Example 3.

N-Phenylacetyl dalargine (5 mg) was dissolved in 2 ml of 0.1 M NaHCO ₃ (containing 50% EtOH) and mixed with 2 ml of a solution of 2-chloroethyl 1-thio-β-D-galactopyranoside (50 mg in 2 ml of 0.1 M NaHCO ₃ ) and the reaction mixture was allowed to incubate at 37 ° C * for 18 hours. After the incubation was completed, the pH of the solution was adjusted to 6.0 by addition of solid NaHCO ₃ . The reaction mixture was separated and analyzed as in Example 1.

and

Example 4.

N-tert-butyloxycarbonyl dalargine (5 mg) was treated as described in Example 3.

Industrial Applicability $ '. 't

_(The substances according to the invention can be used in basic research, in the pharmaceutical industry and in health care.

Claims (2)

Patent claims 1. Glycosylated N-protected analogues of dalargin of formula I, Z I -Tyr-D-Ala-Gly-Phe-Leu-ArgOH CD wherein X is phenylacetyl or tert-butyloxycarbonyl; Y is hydrogen when Z is 4- (O-beta-D-galactopyranosyl) D-deoxy-1-N-aminoglucitol (compounds I, II), or thiogalactosyl when Z is hydrogen (compounds III, IV). Process for the preparation of glycosylated N- protected dalargine analogues according to claim 1, characterized in that the N-protected dalargine analogues are treated with a reducing sugar in the presence of sodium cyanoborohydride (compounds I and II) or chloroethylthiogalactopyranoside (compounds III and IV).