CS229896B1 - Vasopressin analogs and methods for their production - Google Patents

Abstract

The present invention relates to vasopressin analogs of formula I-1-Tyr-Phe-Gln-Asn-Pen-Pro-Lys-Gly-NHg (I) wherein X represents a cysteine or penicillin residue, and a process for their preparation. These analogs are characterized by having an inhibitory (antagonistic) effect on some biological activities of neurohypophysic hormones. The principle of the process for the production of vasopressin analogues is that linear peptides of the formula IX H H 1 -Tyr-Phe-Gln-Asn-Pen-Pro-acid-Gly-NHg (II) wherein X has the same meaning as in formula I containing! two sulfhydryl groups, oxidize to form a disulfide bond in a known manner.

Description

The present invention provides vesopressin analogues with cysteine residues at position 6 ev. at positions 1 and 6 changed for penicillamine (Pen) residues.

These are analogues with inhibitory effects on some biological activities of neurohypophysic hormones. These inhibitors can serve both to detect non-wohypooysary hormones in body fluids and from the point of view of potential medical conditions, i. in pathological overproduction of endogenous hormones. ·

We have previously found (cf. AO 229 893) that lysine-vasopressin analogs having a penicillamine residue at position 1 instead of a cysteine residue have marked inhibitory effects. We have now found that vasopressin analogs in which the remainder of the penicillamine is at position 6 and at positions 1 and 6 have potent inhibitory properties.

The present invention provides novel vasopressin analogs of formula (I)

X-Tyr-Phe-Gln-Asn-Pen-Pro-Lys-Gly-NHg (I) * wherein all amino acids are L-configurations and X is either a cysteine residue or a penicillamine residue.

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

Xη

X-Tyr-Phe-Gln-Asn-Pen-Pro-Lys-Gly-N? (II) wherein X has the same meaning as in Formula I, oxidizes to form the Sisulfide bond ‘.

Some inhibitory effects of the vasopressin analogs of the invention are shown in Table 1.

Table I

PAg values of [S-lysine] vasopressin analogs

Plug Antivasopressor effect k ^{a, c} Antioxytoxický ^ú dumbbells ^Q b β in vitro, in vivo I, X = Cys 6.73 6.00 6.16 I, X = Pen 7.18 6.86 6.80

? The pA _{2 value} is defined as the negative logarithm of the inhibitor concentration that reduces the response of 2X agonist units to equal the response of 1X agonist units in the absence of? ^pAg value is fofinovtaa I ^{OZ stork} arable logarithm of the inhibition constant according to the formula PA = -log75 --- 77 --- r where B is the concentration irhibitoru ^kA * 50B ^/ 50 ^A; (M Ajq is the concentration of agonist producing 50% of meacimálního effect (M) and N, Qg is the concentration of agonist producing 50% of maximum effect of the inhibitor present oti Β (M). ^^ Libice [e ^ s ^ nu ⁱ Jvjstpree Ojytocin.

The process for producing vasopressin analogs is further illustrated in the Examples

Methods used in the examples:

Amino acid analyzes were performed on an automatic instrument (Development Workshops of the Czechoslovak Academy of Sciences, type 6020). The samples were hydrolyzed with 6M-HCl under a vacuum of 150 Pa at 105 ° C for 20 hours. The oxidation was performed with permetic acid. Thin layer chromatography was performed on silica gel plates (Silufol, Kavalier) in systems; 1-butanol-pyridine-acetic acid-water (15: 10: 3: 6) (S4) and chloroform-methanol-acetic acid-water (15: 10: 3: 2) (S6).

Analytical electrophoresis was performed on Whatman 3MM paper in a humid chamber at a potential gradient of 20V / cm for 1 hour in 1M acetic acid (pH 2.4) and pyridine acetate buffer (pH 5.7). The substances were detected by ninhydrin or a chlorination method. Preparative non-carrier electrophoresis was performed on a previously described apparatus (Prusik Z., Sedlakova E., Barth T. Hoppe-Seyler's Z. Physiol. Chem. 353. 1837 (1972)).

EXAMPLE 1 [α-Penicillamine, 8-Lysine] Vasopressin

The protected nonapeptide amide, S-benzyloxycarbonyl-benzyleysteinyl-tyrosyl-L-phenylalanyl-glutamyl-aspartyl-S-benzylpenicilaminyl-propyl-N-p ^£ toluensulfonyllysyl-glycine (90 mg), prepared by stepwise construction of the peptide chain in solution was reduced with sodium in liquid ammonia (100 mL) until the reaction mixture turned blue for 20 seconds. The solution was destained with ammonium chloride, and the ammonia was de-lyophilized. The residue was dissolved in 0.01M HCl (100 mL), diluted with water (50 mL) and shaken with ether. After dilution to a volume of 300 ml, the pH of the solution was adjusted to 7.0 and oxidation was performed by adding 3.3x10 &^lt; ^{2 >} MK < ^{4 >} Fe (CN) g to a steady yellow color for 1 hour. to 7.0.

After the oxidation was complete, the reaction mixture was acidified with acetic acid to pH 4.5 and loaded onto a carboxylate cation exchange column. The column was washed with 0.25% acetic acid and the product was washed with 50% acetic acid. After lyophilization, the product was purified by non-carrier continuous electrophoresis. 10.2 mg of lyophilisate of [α] D -13.6 ° (c 0.13, 3M acetic acid) was obtained. Eg ¹ ® ° · ⁶⁶ . ^E 57 °> ⁶¹ ° · f ^{r> 33} <S4) °. ⁰³ (S6). Amino Acid Analysis: CysCO 3 H + + Pen (O 3 H) (1.94), Tyr 0.92 (0.56), Phe 1.00 (0.96), Olu 0.92 (0.91), Asp 1 .00 (1.01), For 1.00 (1.03), I ss 1.07 (0.98), Gly 0.96 (1.07). Values for the sample oxidized with permic acid are given in brackets. For Ο ^ Ν θΗ ^^ ^^ O ₁₂ S ₂ »^ COOH.8H 5CH ₂ O (1529) calculated: 41.81% C, 6.35% H, 10.93% N;

Found: (corrected for 9% ash) 41.50% C, 6.44% H, 10.92% N.

Example 2 [1-Penicillamine Disulfide, 6-Penicillamine, 8-Lysine] Vasopressin

The protected nonapeptide, benzyloxycarbonyl-S-benzylpenicilaminyl-tyrosyl-phenylalamyl-glutaminyl-asparaginyl-S-benzylpenicilaminyl-prolyl-N ^E- p-toluenesulfonyllysyl-glycine amide (120 mg), prepared by sequential construction of the peptide chain in solution, was reduced , oxidized with potassium ferricyanide and purified as in Example 1. 10.5 mg of the product were obtained at [α] -11.0 ° (c 0, 13, 3M acetic acid), Eg ¹ ® 0.61.0, 60; Ry 0.36 (S4), 0.04 (S6). Amino acid analysis (values in brackets for the sample oxidized with permic acid): Pen (O 2 H) (1.60), Tyr 0.66 (0.82), Phe 1.00 (1.06), Olu 0, 92 (0.98), Asp 1.00 (0.91), Pro 1.00 (1.13), Lys 1.00 (0.98), Oly 1.00 (0.99). For the ^C ₅ ^H 73 ^N ° i3 i2 ^S 2 · ^{3 '5} CH.jCOOH.5HgO (1443) calculated: 48.57% C, 6.92% H, 12.89 "N;

Found: C, 48.54; H, 6.80; N, 12.86.

Claims (2)

A. Vasopressin analogs of formula I X-Tyr-Phe-Gln-Aen-Pen-Pro-ty-Gly-1Wg (I) wherein all amino acids are L-configurations and X destroys the cysteine or penicillamine residue. and 2. A process for the preparation of the novel vasopressin analogs of formula (I) according to claim 1, characterized in that the ee is a linear peptide of formula (II). Η H * ^{XxTyr-Phe-Gln-A} 8N pén-? Ra ro- ^Iy-Gly-L 2 ^H in which X has the same meaning as · in formula I, is oxidized to form a disulfide bond.