CS270051B1 - Oxytocin analogues - Google Patents

Abstract

The solution concerns new oxytocin analogues of formula II Me \ CH -CH -CO-Tyr-Ile-Gln-Asn-Cys-X-Leu-GlyNH , where X denotes (3,4 )dehydroproline (Ila), or 3,4- C3H2) proline (Hb) and the chloral amino acids have the L configuration. Analog Ha is prepared by building the peptide on a solid phase and its cleavage using liquid hydrogen fluoride. Analog lib is prepared by catalytic reduction of substance Ha with gaseous tritium. Substance Ila serves as a precursor of substance lib, which is used in the study of the distribution and metabolism of the clinically used substance I (X-Pro).

Description

The present invention relates to synthetic analogs of oxytocin, a natural peptide hormone from the posterior lobe of the pituitary gland. , USA, 1987). Among the oxytocin analogs used in practice for beneficial properties was also [2-O-methyltyrosine] deamino-1-carbaxytocin (author's certificate No. 149o28) formula I, which for reduced side effects and prolongation of the effect on uterine contractions and milk excretion (AO 167829) was put into use in veterinary practice. For the expansion of indication areas, as well as the possible development of new analogues is hindered by little knowledge about its metabolism and distribution in animal tissues and fluids after its administration to the body. One of the effective ways of understanding metabolism and distribution is the use of a suitable labeled substance. Its metabolic products and changes in the levels of the intact substance can be monitored more appropriately than the unlabelled substances themselves.

Me \

CH ₂ -CH ₂ -CO-Tyr-ne-Gln-Asn-Cye-Pro-beu-GlyNH ₂ -------------------------- -------------------------------------------------- ----------------- ¹ (I)

[2-O-methyltyroain J to amino-1-carboxyxytocin

The present invention relates to two novel analogs of β-O-methyltyrosine deamino-1-carbooxytocin, namely [2-O-methyltyrosine, 7- (3,4) dehydroproyl] deamino-1-carbonyloxytocin (Formula IIa) and [2-O-Mothyltyrosine, 7- (3,4- [ ³ H ₂ ] -prolin)] Deamino-1-carbooxytocin (Formula IIb) and a process for their preparation. In the following, the preparation of both substances and their characteristics are illustrated by examples.

N ^ e.

CH_-CH_ — CO-Tyr-Ile-Gln-Asn-Cys — X — Leu-GlyNH_

I ^{2 2} _______________________________________ | ² ch ₂ ------------------------------- ¹ (n)

Ila, X - (3,4) dehydroproline | lib, X - 3,4-C ^ H ^ J-proline

Example 1

The synthesis of [2-Q-methyltyrosine, 7- (3,4) dehydroproline] deamino-1-carbooxytocin was performed on p-methylbenzhydrylamine resin (0.4 g, 0.8 mmol NH 4/g). Following carboxyterminal amino acid binding (Boc-Gly), Boc-Leu, Boc-A '' -Pro, FmocCys (C listed in Table 1. After addition of the last amino acid, the Boc-protecting group was cleaved with trifluoroacetic acid, the resin was neutralized with a solution of triothylamine, and the peptide was cyclized by 10 equivalents of dicyclohexylcarbodiimide and N-hydroxybenztriazole in 2,2,2-trifluoroethanol. After obtaining a negative result in the ninhydrin test, the resin was washed thoroughly with dimethylformamide and dichloromethane. The resin and bound peptide were dried in vacuo, depleted in a Teflon apparatus · 20 ml of liquid hydrogen fluoride, 1 ml of anisole and 0.5 ml of ethanedithiol. After stirring for 1 h at 0 ° C, the hydrogen fluoride was evaporated, the suspension was washed with ethyl acetate on a frit and after drying in vacuo the product was eluted with 30% acetic acid. The obtained solution was lyophilized.

Example 2

A portion (50 mg) of the lyophilisate was dissolved in 3 mol / L (4 mL) of AcOH and applied to a Bio-Gel P-4 column (7 oxl cm). Fractions containing the desired product were pooled and lyophilized. Product

CS 270051 B1 was dissolved in 3 ml / 1 AcOH 2 ml and applied to a Separon SI C-18 column (25 x 0.4 cm). Eluting with 50% methanol in 0.55% trifluoroacetic acid, the fraction containing 2-O-methyltyroline, 7- (3,4) dehydroproline deamino-1-carbooxytocin was concentrated in vacuo and lyophilized. 6.2 mg of a chromatographically uniform product are obtained, the biological properties of which are given in Table 2, which is converted into [3-O-methyl-tyrosine] by hydrogenation on freshly reduced palladium in dimethyl acetate. deamino-1-carba-oxytocin (comparison by HPLC and biological activities).

Table 1

Construction of the peptide chain on the resin using Fmoc-amino acid or Boc-amino acid. General scheme.

Fmoc -amino acids Boc-amino acids step reagents (15 ml) min step reagents (15 ml) min 1 CH ₂ C1 ₂ (2x) 3 1 CH ₂ C1 ₂ (4x) 7 2 DMF (2x) 3 2 45% TFA V CH ₂ Cl ₂ 2 3 DMFtpiperidine (1: 1) (2x) 2o 3 _ _ 2o 4 DMF (2x) 3 4 CH ₂ C1 ₂ (3x) 5 5 dioxane: H ₂ O (2 t 1) (2x) lo 5 8% TEA in CH ₂ C1 ₂ (2x) 3 6 DMF (3x) 5 6 CH ₂ C1 ₂ (4x) 7 7 CH ₂ C1 ₂ (or DMF) (3x) 5 7 HOBt ester Boc-Ak art symmetric anhydride (3 eq.) V ch ₂ ci ₂ 4o or more 8 HOBt ester Fmoc AK 21 symmetrical anhydride (3 eq.) In CH ₂ C1 ₂ art. CH ₂ C1 ₂ : DMF 15 and more 8 ninhydrin test 5 9 ninhydrin test 5 9 CH ₂ C1 ₂ (3x) 5 lo repeat condensation if necessary (steps 7 to 9) lo 2-propanol (3x) 5 11 DMF (5x) 8 11 CH ₂ C1 ₂ (4x) 7 12 1-propanol (5x) 8 12 repeat condensation if necessary (steps 7 to 11) 13 CH ₂ C1 ₂ (5x) 8

Note

TEA

AK

DME trlethylamine amino acids and dimethylformamide

CS 27oo51 Bl

Table 2

Biological properties of t2-O-methyltyrosine] deamino-1-carboxyoxylone and a derivative with 3,4 dehydroproline in position 7

Substance

Rat uterus

Gaiactogoglic test in vitro in vivo in vivo (intensity of effect.

IU / mg

IU / mg

IU / mg)

C 2 -O-Methyltyrosin-3-17 deamino-1-carbooxytocin

17.1

[2-O-Methyltyrosine, 7- (3,4)

36.2

169.3

91, or de proline-deamino-1-carbooxytocin

Example 3

19 mg of PdO was pre-reduced with H 2 in methanol, which was subsequently evaporated. To the catalyst were added 0.5 mg of [2-O-methyl tyrosine, 7- (3,4) dehydroproline-3-deamino-1-carboxyxycin] and 1 ml of dimethylacstamide. The mixture was reduced with 60% gaseous gas for 2.5 h at a pressure of 84 kPa.

After lyophilization with DMAA and centrifugation of the catalyst, the product was isolated by HPLC on S paron SX 18, 25o x 8 mm with 53% aqueous methanol. The yield was 15 MBqq. A biological test on an isolated rat cannon * in situ showed that a total of 15o AS (0.15 βmol) of biologically active triclated C2-O-methyltyrosineJdeamino-1-carbooxytocin was obtained, the specific reactivity of which was (0.99 TBq / mmol).

Claims (2)

OF THE INVENTION Oxytocin analogues of the general model · 11. ^Me . CH _o -CH „-CO- \ ryr — Ue — Gin — Asn-Cys — X — Leu — GlyNH, • ZZ। Z wherein X is (3,4) dehydroproline or 3,4-C ³ H ₂ proline and chiral amino acids have the L configuration.