CS230315B1 - Vasopressin analogs and methods for their production - Google Patents

Abstract

The invention relates to vasopressin analogues of the chemical formula 2 Cys—Tyr—Phe—Gln—Asn—Cys—Pro— (I) where X is D-arginine or ornithine and a method for their production. These vasopressin analogues, having a non-protein basic amino acid in position 8 and, in comparison with the natural hormone, the glycinamide residue removed from positions 1/9, retain their effect on the central nervous system with very low endocrine activity. The essence of the method for the production of these analogues lies in the fact that linear peptides of the chemical formula II Η H Cys-Tyr-Phe-Gln-Asn-Cys-Pro- (II) where X has the same meaning as stated above, are oxidized to form a disulfide bond.

Description

(54) Vasopressin analogues and method of their production

The invention relates to vasopressin analogues of the chemical formula

Cys—Tyr—Phe—Gin—Asn—Cys—Pro— (I) where

X stands for D-arginine or ornithine and the method of their production.

These vasopressin analogues, having a non-protein basic amino acid at position 8 and, compared to the natural hormone, the glycinamide residue removed from positions 1/9, retain their effect on the central nervous system with very low endocrine activity.

The essence of the method of producing these analogues is that linear peptides of chemical formula II

H H

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-(II) where

X has the same meaning as above, are oxidized to form a disulfide bond.

230313

The invention relates to vasopressin analogues with a non-protein basic amino acid at position 8 and with the glycinamide residue removed at position 9, and a method for their production. These are substances that have significant effects on the central nervous system.

It is known that neurohypophyseal hormones and some of their analogues affect memory, sleep and learning in experimental animals. However, it is necessary that the substances used do not have the endocrine activities of natural hormones, which are highly undesirable, especially for substances of the vasopressin type (antidiuretic and pressor activity) (cf. Czechoslovak author's certificate number 217 633).

It has now been found that a suitable dissociation of activities can be achieved by introducing a non-proteinaceous, basic amino acid into position 8 of the vasopressin molecule, while simultaneously removing the glycinamide residue from position 9.

The invention relates to vasopressin analogues having the general formula I where all amino acids are L-series and

X denotes D-arginine or ornithine (a non-protein, basic amino acid of the L or D series).

The essence of the method for producing new vasopressin analogues of formula I according to the invention is that the linear peptide of formula II

H H

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-(II) where

X has the same meaning as above, is oxidized to form a disulfide bond in a known manner.

Some endocrine biological effects are listed in Table 1.

Cys—Tyr—Phe—Gin—Asn—Cys—Pro— (I)

TABLE 1

Substance Biological activity ^and

Uterotonic Galactogogic Pressoric Antidiuretic anesthetize- neanestheti- van used rat rat I, X = D-Arg 0.07 0.12 < 0.2 4.5 0.004 ^points I, X = Orn 0.09 0.02 < 0.2 0.29 0.001 ^points

^a Determined in rats and expressed in international units per 1 mg; ^b expressed as % of the effect of [8-D-arginine]deamino-vasopressin (DDAVP) at the threshold level.

The prepared analogues were tested in the passive defense reaction test, the so-called passive avoidance, in which the beneficial effects of natural vasopressin on memory processes were demonstrated. The essence of the test is that rats avoid the area in which they received a mild electric shock to the paws. The duration of this avoidance is evaluated. If the administered substance prolongs this reaction, the effect can be interpreted as strengthening the memory trace related to this experimental situation. The substances were administered at a dose of 5 µg. . kg” ¹ subcutaneously either immediately after the animals received the shock to the paws or 20 hours before the avoidance reaction retention test. The fact that the effect was still evident 20 hours after application indicates a long-term effect of the analogues, which is very desirable in this regard.

The method of producing vasopressin analogs is further illustrated in the examples. Methods used in the examples:

Amino acid analyses were performed on an automatic two-column apparatus (Development Workshops, Czechoslovak Academy of Sciences, type 6020). Peptide samples were hydrolyzed for 20 hours in 6M-HCL at 105 ^° C in a vacuum of 160 Pa. Thin-layer chromatography was performed on silica gel plates (Silufol, Kavalier) in the following systems:

Sl: 2-butanol — 98% HCOOH — HaO (75 : 13.5 : 11.5),

S2: 2-butanol — 25% NHOH — HzO (85 : 7.5 : 7.5),

S3: 1-butanol — CHsCOOH — H2O (40 : 10 : 10),

S4: 1-butanol — CH3COOH — pyridine — — H2O (15 : 3 : 10:6),

S13: 1-butanol — CH3COOH — H2O (50:15:40),

S23: ethyl acetate — pyridine — CHsCOOH — — H2O (5:5:1:3).

Electronic analysis was performed on Whatman 3MM paper in a humid chamber at a potential gradient of 20 V/cm. Substances were detected by ninhydrin or chlorination method.

Example 1

N-benzyloxycarbonyl-S-(2,4,6-trimethylbenzyl)cysteinyl-tyrosyl-phenylalanyl-glutaminyl-asparaginyl-S-(2,4,6-trimethylbenzyl)cysteine 1-proly lN ⁵ -benzyloxycarbonylornithine benzyl ester

To a solution of N-benzyloxycarbonyl-S-(2,4,6-trimethylbenzyl)cysteinyl-tyrosyl-phenylalanyl-glutamyl-asparaginyl-S-(2,4,6-trimethylbenzyl)cysteine hydrazide (238 mg) in dimethylformamide (2 ml) was added 3M-HCl in dioxane (130 μΐ) with stirring, the solution was cooled to — 20 °C and butyl nitrite (21 mg) in dimethylformamide (0.5 ml) was added. The reaction mixture was stirred for 20 minutes, cooled to —40 °C, neutralized with N-ethylpiperidine (pH 7, wet pH paper) and a solution prepared as follows was added:

Benzyl ester of o-nitrobenzenesulfenylprolyl-N5-benzyloxycarbonylornithine (182ml) was dissolved in dimethylformamide (1ml) and 3M-HCl in ether (0.5ml) was added to the solution. After 5 min. the reaction mixture was diluted with ether, the precipitate was isolated by decantation and washed with ether. The hydrochloride obtained was dissolved in dimethylformamide (2ml), the pH of the solution was adjusted to 10 with N-ethylpiperidine and the solution thus obtained was added to the azide solution, the preparation of which is described above. After 60 hours at 0°C, the reaction mixture was evaporated, the residue was triturated successively with 0.5M-HCl, water, saturated NaHCO3 solution and again with water. The product was purified by crystallization from a dimethylformamide-water mixture and gel filtration in dimethylformamide. 300 mg (93%) of product were obtained, mp 238-240°C, [α] _D -27.6° (c 0.15, dimethylformamide). R _f 0.86, (S1), 0.67 (S2), 0.82 (S3), 0.91 (S4).

For C86H103N11O16S2.2H2O (1617) calculated:

63.88% C, 6.67% H, 9.53% N, 3.97% S; found:

64.00% C, 6.38% H, 9.57% N, 3.95% S.

The above-described protected octapeptide (100 milligrams) was dissolved in trifluoroacetic acid (1.25 ml), thioanisole (100 μΐ) was added to the solution, and the solution was cooled to 0 ° C. Trifluoromethanesulfonic acid (1 ml) cooled to the same temperature was added to the reaction mixture. After 30 minutes at 0 ^° C, the mixture was diluted with ether, the precipitate was filtered off and the filtered portion was dissolved in water (300 ml). The pH of the solution was adjusted to 6.8 with 0.1H-NaOH and the reaction mixture was oxidized with atmospheric oxygen for one hour. The pH was adjusted to 3.9 with acetic acid and the solution was filtered through a weakly basic ion exchange column in an acetate cycle. The effluents were lyophilized (89 mg) and the lyophilizate dissolved in 50% aqueous acetic acid and the product was purified by gel filtration. 15.3 mg (24% of the compound was obtained with [α]D — —15.1° (c 0.2, 1M acetic acid); E24 ^GIy 0.89, E57 ^His 0.34; R _F 0.37 (S4), 0.43 (S13), 0.89 (S23).

Amino acid analysis:

Phe 1.01, Tyr 0.92, Glu 1.00, Asp 1.00, Pro 1.03, Orm 0.99. The amount of cystine was determined in a separate sample after oxidation with performic acid and was determined as cysteic acid: 1.92.

For C45H59N1O12S2.2 CH3CO2H.2 H2O (1142) calculated:

49.42% C, 6.26% H, 13.48% N;

found:

49.18% C, 5.95% H, 13.30% N.

Example 2

N-Benzyloxycarbonyl-S-(2,4,6-trimethylbenzyl)cysteinyl-tyrosyl-phenylalanyl-glutaminyl-aspar aginyl-S-(2,4,6-trimethylbenzyl)cysteine 1-propyl-N ^G -p-toluenesulfonyl-D-arginine benzyl ester

The azide protected hexapeptide solution was prepared in the same manner as described in Example 1.

Benzyl ester of o-nitrobenzenesulfenylprolyl-N ^G -p-toluenesulfonyl-D-arginine was prepared according to Czechoslovak author's certificate number 217 633. To a solution of this substance (200 mg) in dimethylformamide (1 ml) was added 2 M-HCl in ether (0.5 ml). After 5 minutes at room temperature, the reaction mixture was diluted with ether, the precipitated hydrochloride was filtered off with suction, washed with ether, dissolved in dimethylformamide (2 ml) and the pH of the solution was adjusted to 10 with N-ethylpiperidine. This solution was added to the azide solution and the reaction mixture was left at 0 °C for 60 hours. The dimethylformamide was evaporated, the residue was worked up and purified in the same manner as in Example 1. 310 mg (93 percent) of product were obtained as a white solid, mp 230-232°C, [.a] _D -28.6° (c 0.5, dimethylformamide). Rf, 0.94 (S1), 0.75 (S2), 0.90 (S3), 0.96 (S4). For C8SH105N13O16S3 . H2O (1961) calculated:

61.08% C, 6.26% H, 10.77% N, 5.69% S; found *

60.89% C, 6.23% H, 10.57% N, 5.42% S.

[8-D-Arginine, 9-desglycinamide] vasopressin

The above-described protected octapeptide (100 mg) was deprotected and oxidized in the same manner as described in Example 1. Desalting and gel filtration were also performed in a similar manner. 14 mg (23%) of the product was obtained with [α] _D — —17.7° (c 0.27, 1M acetic acid); E ₂ ? ^Iis 0.56, E57 ^His 0.32; RF 0.34 (S4), 0.42 (S13), 0.91 (S23).

Amino acid analysis:

Phe 1.02, Tyr 0.91, Asp 1.02, Glu 1.04, Pro

1.02, Arg 1.00; Cys (O3H) 1.92 (the value for cysteic acid was obtained in a separate sample after oxidation with performic acid).

For C41H6iN13O12S2.2CH3CO2H.2H2O (1124) calculated:

49.14% C, 6.19% H, 16.20% N; found:

49.02 θ/o C, 5.98% H, 15.94% N.

Claims (2)

Subject Vasopressin analogs of formula I Cys-Tyr-Phe-Gin-Asn-Cys-Pro- (I) wherein all amino acids are L-series and X is D-arginine or ornithine. vynalezu 2. A process for producing novel vasopressin analogs of formula (I), characterized in that the linear peptide of formula (II) Η H I I Cys-Tyr-Phe-Gln-Asn-Cys-Pro- (II) wherein X has the same meaning as above, is oxidized to form a disulfide bond.