CS231749B1 - Analogy of vasopresine and its processing method - Google Patents

Description

The present invention provides a vasopressin analogue and a process for its preparation. It is an analogue stabilized to enzymatic cleavage and with the glycinamide residue removed at the 9 position, which has significant central nervous system effects.

Neurohypophyseal hormones and some of their analogues are known to affect memory, sleep and learning in experimental animals (D. de Wied: Proc. Roy. Soc. B210. 183/1980) ’. For practical use, however, it is essential that the substances used do not have the endocrine activities of natural hormones (cf. α1,27633; α.230,315), and that they are protected against enzymatic cleavage (Hauzer K., Barth T .: Chem. Sheets 74. 1029 / 1980).

It has now been found that an analog of the present invention, which has a glycinamide residue at the 9-position removed compared to the natural hormone, is replaced by the L-arginine at position 8 with the stereoisomeric form and the disulfide bond with the thio-ether bond.

The present invention provides a vasopressin analog of Formula I

CH-CH-S-CH ² _O 2, 2

CHg-CO-Tyr-Phe-Gln-Asn-NH-CH-CO-Pro-D-Arg (I) wherein all amino acids are L-series except arginine, which has a D configuration. , The Gin residue of glutamine, the Asn residue of asparagine, the Pro residue of proline, and the Arg residue of arginine.

The process for the preparation of the novel vasopressin analog of the formula I according to the invention is based on the preparation of a peptide of the formula II

CH ² -CH ² -S-CH, ^{2 2} t ²

CH ₂ -CO-Tyr-Phe-Gln-A sn-NH-CH-CO-Pro-Arg / X / -Y (II) wherein all amino acids are L-series except arginine having the D configuration and wherein X indicates the side chain protecting group of arginine and the Y protecting group of the carboxy group of arginine, cleaving these protecting groups in a known manner.

The analog of Formula I inhibited the lysinvasopressin presoric activity in a despinalized rat assay. The anti-diuretic activity determined in the anesthetized rat was 0.8 IU / mg. In a non-anesthetized rat assay, Compound I had an anti-diuretic activity 4.5 to 5 orders of magnitude lower than the corresponding analogue in which the glycinamide residue is retained (oo 171787); this figure is determined on the basis of equipotent doses (T, / 2 ^{= 200} minutes) Škopková J., Hrbas P., Slaninová J., Barth T .: Collection Czechoslov. Chem. Commun. 46, 1850 (1981). The effect on the central nervous system has been tested in a passive defense response (passive avoidance) test. In this test, substance I according to the invention appeared to be more advantageous than the substances previously described (and 217633, and 230315).

The process for the preparation of the analogue according to the invention is illustrated in the example.

Example

Preparation of starting compounds (intermediates). ?

Preparation of o-nitrobenzenesulfenylprolyl-N-p-toluenesulfonyl-D-arginine benzyl ester.

A solution of 2,4,5-trichlorophenyl-nitrobenzensulfeny proline (2.5 g) and the hydrobromide of N ^G -p-toluenesulfonyl-D-arginine (2.5 g) in DMF (5 mL) was stirred for 40 h at r.t. . The dimethylformamide was evaporated in vacuo, the residue dissolved in ethyl acetate, the ethyl acetate solution was extracted with saturated sodium bicarbonate solution, water, KHSO4 / H2SO4 solution pH 2 and water. After drying over MgSO 4, evaporation of ethyl acetate and crystallization from ethyl acetate and petroleum ether, 3 g (90%) of the product were obtained, mp 90-92 ° C, DG _B -40.4 ° (c 0.4 methanol); Hy 0.88 (S1), 0.75 (S2), 0.75 (S3), 0.83 (S4),

0.60 (S9). For C ₃₁ H _{36 6 6 7} S ₂ (668.8) calculated: 55.67% C, 5.43% H, 12.56% N, 9.59% S; Found: 55.92 »C, 5.38% H, 12.68% JS, 9.38% S.

Preparation of benzyl lactam tyrosyl-phenylalanyl-glutaminyl-asparaginyl-S / _Y carboxypropyl / cysteiioyl-prolyl-N ^G -p-toluenesulfonyl arginine-B (compound of formula II).

To a solution of the protected benzyl ester described above (177 mg) in dimethylformamide (1 mL) was added 2.6 M hydrogen chloride in ether (1 mL) and the reaction mixture was left for 4 min. at room temperature, the hydrochloride formed was precipitated with ether and dried (Eg ¹ = = 0.96, Ε γ = 0.70).

To a solution of the hydrochloride in dimethylformamide (1 mL) was added N-ethylpiperidine to a pH of about 10, and a solution of 1-deamino-1-carbo-pressinic acid was added (Brtnik i ', Barth T., Jost K. (Collect. Czechoslovak. Chem. Commun. £ 6, 278 (1981)) (100 mg) and N-hydroxybenztriazole (23 mg) in dimethylformamide (1.5 mL). The reaction mixture was cooled to -30 ° C, dicyclohexylcarbodiimide (31 mg) in dimethylformamide (0.5 mL) was added and the mixture was stirred for 4 h at -5 ° C and 20 h at room temperature. The dicyclohexylurea was filtered off, the dimethylformamide was evaporated, the residue was rubbed with hydrochloric acid (pH 2), washed on the filter with water, saturated sodium bicarbonate solution, water and ether. 160 mg of crude product was obtained, which was purified by gel filtration in dimethylformamide. The effluents containing the pure material were evaporated and the residue was crystallized from dimethylformamide and water. Yield: 100 mg (60%) m.p. 152 DEG-154 DEG C. [.delta. 0.50 (S1), 0.54 (S3), 0.66 (S4) · Amino acid analysis:

For 0.98, Arg (0.97, Cys / C ^CO COH) 0.94, Glu 1.05, λ _s 1.04, Tyr 0.92, Phe 1.08,

For C ₅₉ H ₇₄ N ₁₂ O ₁₄ S ₂ . HgO (1257) calculated: C 56.36, H 6.09, H 13.37, S 5.10; Found: C 56.52, H 6.13, N 13.27, S 5.09.

Preparation of the final product

Lactam tyrosyl-phenylalanyl-glutaminyl-asparaginyl-S - (- carboxypropyl) cysteinyl-prolyl-D-arginine (compound of formula I)

A solution of the compound of formula II (30 mg) in trifluoroacetic acid (300 µl) was cooled to 0 ° C, trifluoromethanesulfonic acid (200 µl) was added and thioanisole (20 ml) was left at the same temperature for 30 min. The reaction mixture was precipitated with ether, the crude free lactam was filtered off, washed with ether and filtered through an anion exchange column in an acetic cycle. The effluents were lyophilized and the lyophilisate purified by non-carrier electrophoresis (2500 V, 135 mA). 6 mg of the product were obtained at [α] _D - 47 ° (c 0.1 1M acetic acid); _Rp 0.34 (S1), 0.57 (S4), 0.74 (S23); E ^ °> ⁶⁰ · Amino acid composition: Arg 1.01, Pro 1.04, Glu 1.01, Asp 1.02, Phe 0.97,

Tyr 0.94, Cys (C 18 H 15 O 6 H) 0.98. For ^C 45 ^H 62 ^N 12 ^O 12 ^S · CHOOSE. % H, 6.54;% N, 15.15. Found:% C, 50.68;% H, 6.45;% N, 14.92.

Analytical methods used in the example:

Amino acid analyzes were performed on an automatic instrument (Development Workshops, Czechoslovak Academy of Sciences, tp. 6020). Peptide samples were hydrolyzed for 20 hours in 6K-HCl at 105 ° C under a vacuum of 150 Pa. Thin layer chromatography was performed on silica gel plates (Silufol, Kavalier) in the following systems:

: 2-butanol-98% HCOOH-HgO (75: 13.5: 11.5): 2-butanol-2 5% NH 4 OH-HgO (85: 7.5: 7.5): 1-butanol- CH 2 OH - HgO (40: 10: 10): 1-butanol - CH 3 COOH - pyridine - HgO (15 = 3: 10: 6)

Ξ9: Benzene with 20% methanol

S23: Ethyl acetate - pyridine - CH 2 COOH - HgO (5: 5: 1: 3)

Electrophoretic analysis was performed on Whatman 3MM paper in a humid chamber at a potential drop of 20 V / cm. The substances were detected by ninhydrin or chlorination method.

Claims (2)

SUBJECT OF THE INVENTION A vasopressin analog of formula I CHó -OHs -CH ~ “2 2 ®, 2 L I CHg-CO-Tyr-Phe-Gln-Asn-NH-CH-CO-Pro-D-Arg (I) wherein all amino acids are L-series except arginine having the D configuration. 2. A process for the preparation of a novel analog of vasopressin of formula I according to claim 1, characterized in that the CHr CH 3 —S-6H _? t CH ₂ -CO-Tyr-Phe-Gln-Asn-NH-CH-CO-Pro-D-Arg / X / -Y (II) wherein all amino acids are L-series except arginine having the D configuration and wherein X denotes a side chain protecting group of arginine and a Y protecting group of a carboxyl group of arginine, cleaving these protecting groups.