CS229895B1 - Analogy of oxytocine and method of its production - Google Patents

Abstract

The invention relates to a novel oxytocin analogue of Formula I Gly-Gly-Gly-Cys-D-Phe (Et) -Ile-Gln-Asn-Cya- -Pro-Leu-Gly-NHg (AND) where all chiral amino acids are L configuration except p-ethylphenylenine (Phe (Et)), which has a D configuration. This oxytocin analogue is elongated uterotonic inhibitory effect oxytocin activity in the uterine test in vivo. The nature of the oxytocin analog preparation method Formula I is that ee is L2-D-p-ethylphenylalanine oxytodn (whose production method is described in PV 3340-82) acylates the active ■ o-nitrobenzenesulfenylglycylglyeyl ester - glycine, preferably with N-hydroxyeukcinimide ester, and o-nitrobenzene sulfenyl a protecting group is spun by action hydrogen chloride in an organic solvent, or with the linear peptide of formula II Η H I I Gly-Gly-Gly-Cys-D-Phe (Et) -Ile-Gln-Asn-Cya- -Pro-Leu-Gly-NH 2 the sulfide bond formation is known to be oxidized way.

Description

The present invention relates to a novel oxytocin analogue of formula I

Gly-Gly-Gly-Cys-D-Phe (Et) -Ile-Gln-Asn-Cya-Pro-Leu-Gly-NHg (I) wherein all chiral amino acids are L configurations except p-ethylphenylalenine / Phe (Et) /, which has a D configuration.

This oxytocin analog exhibits a prolonged inhibitory effect on the uterotonic activity of oxytocin in the uterine test in vivo.

The principle of the process for the preparation of the oxytocin analog of the formula I is characterized in that ee L2-Dp-ethylphenylalanine oxytodium (the production method of which is described in PV 3340-82) is acylated with an active o-nitrobenzenesulphenylglycyl-glyeyl-glycyl ester. the o-nitrobenzene sulfenyl protecting group is removed by treatment with hydrogen chloride in an organic solvent, or the linear peptide of formula II

Η H

I I

Gly-Gly-Gly-Cys-D-Phe (Et) -Ile-Gln-Asn-Cya-Pro-Leu-Gly-NH ₂ is oxidized to form the sulfide bond in a known manner.

It is an object of the present invention to provide an enelog, an oxytocin having a protracted inhibitory effect of the uterotonicity of oxytoein in an in vitro test, and a method for its manufacture.

Oxytoeine analogs having inhibitory effects on the biological activities of oxytocin may find application in medical practice (see, e.g., U.S. Pat. No. AO 226 923 and U.S. Pat. No. 229 893).

Neurohypophyseal hormones, as a substance of peptide character, succumb! in the body by enzymatic conversion (Heuzer K., Berth T .: Chem. sheets 21, 1029 (1980)). This fact has previously been used in the preparation of so-called hormonogens, ie substances from which the active substance is generated in the body, eg by the action of enzymes (Beránková-Ksandrová Z., Bisset O. V.,

Jošt K., Krejčí I., Plieke V., Rudlnger J., Rychlík I., Sorm P .: Brit. J. Phermecol. ££,

615 (1966); Lebl M., Berth T., Jost K .: ColleCt. Czech. Chem. Commun. 43, 1,300 (978)). However, substances which, by enzymatic cleavage, release a hormone inhibitor (i.e. a so-called ogene inhibitor) have not been prepared.

We have now found that by a suitable combination of structural features in the molecule, i. by acylation of the amino-terminal portion with the short peptide chain together with the stereosubstituted aromatic amino acid at position 2, an oxytoeine analogue (Formula I) can be obtained which exhibits a prolonged inhibitory effect on the uterotonic activity of oxytocin in the uterine test in vivo compared to the equipotent dose of predicted generated substance. -D-Phe (Bt)] oxytocin).

The present invention provides a novel oxytocin enelog of formula (I)

Gly-Gly-Gly-Cy 8 -D-Phe (Et) -Ile-01n-Asn-Cye-Pro-Leu-Qly-NH ₂ (I) wherein all chiral amino acids are L configurations except p-ethylphenylalanine / Phe (Et) ) /, which has a D configuration.

The process for the preparation of the novel oxytocin analog of the formula I according to the invention is characterized in that [2-Dp-ethylphenylelenine] oxytocin (production method see AO 226 923) is acylated with an active o-nitrobenzenesulphenylglycyl-glycyl-glycine ester, preferably The nitrobenzenesulfenyl protecting group is removed by treatment with hydrogen chloride in an organic solvent. Another possible process for the preparation of the compound (I) according to the invention is by using a linear peptide of formula (II)

Η H

Gly-Gly-Gly-Cyya-D-Phe (Et 1 -Ile-Gln-Asn-Cys-Pro-Leu-Qly-NH 3 (II) oxidizes to form a disulfide bond.

The production method is further illustrated in the examples.

Example 1

To a solution of [2-D-p-ethylphenylalenine] oxytocin (4 mg) in 0.3 mL of water was added a solution of N-hydroxysuccinimide ester of o-nitrobenzenesulfenylglycyl-glycyl-glycine (1.0 mg) in 0.3 mL of dimethylformamide. After stirring at room temperature for 2 h, 4 mg of active triglycine ester was added and the solution was made alkaline by addition of 1 M NaOH to pH 8. The reaction was monitored by reverse-phase high-pressure liquid chromatography. After disappearance of the starting material (20 h), the solution was evaporated in an oil pump vacuum at room temperature, the residue was dissolved in methanol (ml) and after addition of 1.3 L HCl in ether (60 µL) and after standing for 5 min at room temperature. the solution was evaporated again.

The residue was suspended in 1 ml of 3 M acetic acid, filtered, and the filtrate was loaded onto a 50 x 0.9 cm (octadecyl chain modified content) silica column (5 9 carbon content) by diluting with 5 ml of water. Elution was performed at a flow rate of 4 ml / mln through the following mobile phase program: 0-40 min methanol-0.19 trifluoroacetic acid 40:60, 40-60 min linear gradient to 609 methanol content. The corresponding fractions were concentrated in vacuo and lyophilized.

2.3 mg of Rj was obtained. 0.04 (2-butanol - 98% formic acid - water; 75: 13.5: 11.5) 0.03 (2-butenol - 25% aqueous ammonia - water; 85: 7 (5: 7.5) ), 0.61 (1-butanol - pyridine - acetic acid - water; 15: 10: 3: 6) E ^ ¹ ® = 0.32 and Eg ¹ ^ = 0.68 (wet chamber, 20 V / cm , pyridinium acetate buffer or 1 M acetic acid) Amino Acid Analysis: Asp 0.99, Glu 1.05,

For 0.90, Gly 4.08, Cys 1.29, Ile 0.96, Leu 1.02, Phe (Et) 0.99.

Example 2

The aqueous solution of the compound II obtained after the removal of the protecting groups from the sulfur atoms of the cysteine residues is oxidized, for example, by treatment with an aqueous solution of KFe (CN) g and the aqueous solution of the product is further processed as shown in Example 1. shown in Example 1.

Claims (3)

An oxytocin analog of formula I I ---- 1 Gly-Gly-Gly-Cya-D-PhetEt-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NHg (I) wherein all chiral amino acids are of the L configuration except p-ethylphenylalanine (/ Phe (Et)), which my configuration D. 2. A process for the preparation of an oxytocin analogue of formula (I) according to claim 1, wherein the aa [2-Dp-ethylphenylelanine] oxytooine is acylated with an active o-nitrobenzenesulphenylglycyl-glyoyl-glyoin ester, preferably using an N-hydroxysuccinimide ester. removal by treatment with hydrogen chloride in an organic solvent. 3. A process for the preparation of an oxytocin analog of formula (I) according to claim 1, characterized in that the linear peptide of formula (II) is used Η H Gly-Gly-Gly-Iy-D-Phe (Et) -Ile-Gln-Asn-Eys-Pro-Leu-Gly-NH oxidizes to form a disulfide bond.