CS217633B1 - Vasopressin analogue and method of preparing same - Google Patents

Description

The present invention provides a vasopressin analog, cyclic disulfide / 3-mercaptopropionyltyrosyl-phenylalanyl-glutaminyl-asparaginyl-cysteinyl-prolyl-D-arginine, and a process for its preparation.

It is a substance that has significant effects on the central nervous system.

Neurohypophyseal hormones and some of their analogs have been shown to affect memory, sleep, and learning in experimental animals (see, e.g., Krejci I., Kupkova B., Kasafirek E., Barth T., Jost K .: Effects of Neurohypophyseal Hormone Analogues and Fragments on Learning and Hemory Processes in Behavioral Tests, in the book Neuropeptides and Neural

Si

CH, i

CH 2 CO-Tyr-Phe. -Gin 2

Transmission, Raven Press 1980, p. 301). However, the disadvantages of using these substances are their endocrine effects, such as oxytocic, galactogogic, presorl, antidiuretic and others. Furthermore, it has been shown that chemical modification of neurohypophyseal hormones can reduce or even eliminate some activities. By a suitable chemical modification, a vasopressin analogue was obtained practically free from endocrine activities but with preserved effects on the central nervous system.

The vasopressin analog of the invention has the formula I,

- _p

CH, and ^Λ Sn NH-CH-CO-P> about-Arg-OH m

wherein all amino acids are L-series, only arginine (Arg) is D-series.

The essence of the process for the production of the novel vasopressin analog of the formula I according to the invention is that the linear peptide of the formula II

217B33

SH SH

CH2 CH2

CHa-CO-Tyr-Phe-Gin-Asn-NH-CH-CO-Pro-Arg-OH (II) oxidizes to form a disulfide bond known Some endocrine biological effects are a procedure. listed in Table I.

TABLE I

Substance Biological activity ³

Presorlcká Uterotonická

Oxytocin 3

Vasopresin 450

Analog of formula I 0.01 ⁸ International Units per mg

The effect of the analogue of formula I on central nervous system influence was tested in two experimental settings:

1. In the so-called passive defense reflex test, where rats avoid the area in which they received a slight electrical shock to their paws. The duration of the passive defense reflex is evaluated; prolongation of this reaction is interpreted as a beneficial effect on memory footprint. The analog of formula I was administered at a dose of 5 μ%! / kg subcutaneously either immediately after the animals received a paw shock or 30 min before the avoidance retention test. The retention test was performed 24 or 48 hours after shock. In both cases there was a statistically significant prolongation of the observed reaction.

2. The effect of the analog of Formula I on the short-term working spatial memory of rats in a 24-arm radial maze was tested. There is a piece of food in each arm of this maze that rewards the rat when it enters. If he visits a shoulder twice, he will not find the reward a second time. The purpose of the experiment is to find out how the rat remembers which channels it has already visited and where it can still find food. The experiment has shown that the analog of Formula I improves memory significantly. If a rat without an administered analog of Formula I makes an average of 3.49 errors, then administration of the compound of Formula I (1 µg / 300 g subcutaneously) reduces the incidence of error by 0.82.

The process for producing a vasopressin analog is further illustrated in the exemplary embodiment.

Example

O-benzyl-N ^G nitrobenzensulfenylprolyl toluenesulfonyl-D-arginine

Hydrobromide To a solution of N ^G -p-toluenesulfonyl-D-arginine (2.5 g) in dimethylformamide (5 mL) was added N-ethylpiperidine (0.8 ml) and 2,4,5-trichlorophenyl-nitrobenzensulfenylprolinu of (2 , 24 g) in dimethylformamide (5 mL). After stirring at room temperature for 40 h, dimethylform was galactogogic

500 500

5

0.08 0.01 amide was evaporated, the residue was dissolved in ethyl acetate and this solution was successively shaken with sodium bicarbonate solution, water, KHSO4 / K2SO4 solution (pH 2) and again with water, dried and evaporated. Crystallization three times from ethyl acetate-petroleum ether gave 3 g (90%) of the product, mp 90-92 ° C [α] _D -40.4 ° (c 0.4, methanol).

For C51H36N6O7S2 (668.8) calculated:

55.67% C, 5.43% H, 12.57% N, 9.59 _0/0 S;

found:

C, 55.92; H, 5.38; N, 12.68., S. 9.38.

Benzyl S-benzyl-.beta.-mercaptopropionyl-tyrosyl-L-phenylalanyl-glutamyl-aspartyl-S-benzylcysteinyl-prolyl-N ^G -n-toluenesulfonyl-D-arginine

To the S-benzyl- [3-mercaptopropionyl-tyrosyl-phenylalanyl-glutaminyl-asparaginyl-S-benzylcysteine hydrazide solution [239 mg; Zaoral M., Kol.)., Shorm F .: Collect. Czech. Chem. Commun. 32, 1250 (1967)] in dimethylformamide (3 mL) was added 2H-HCl in dioxane (0.26 mL), the reaction mixture was cooled to -20 ° C and butylnitrite (25.8 mg) in dimethylformamide was added. (0.8 mL). After 20 min. the mixture was cooled to -40 ° C and neutralized with N-ethylpiperidine and the solution was added a solution of benzyl ester hydrochloride prolyl-N ^G -P-toluensulfonylargininu (previously prepared from 250 mg of the protected dipeptide cleavage-nitrobenzensulfenylové protecting group with HCl in ether). After standing at 0 ° C for three days, dimethylformamide was evaporated, the residue was triturated with 1% hydrochloric acid, filtered and washed on the filter with water, saturated NaHCO 3 solution and water. The crude product was purified by gel filtration in dimethylformamide solution. After reprecipitation from dimethylformamide with water, 280 mg (78%) of the product was obtained at mp 174-175 ° C, [α] _D -27.6 (c 0.15, dimethylformamide).

For C72H86N12O14S3.0.5 H2O (1148) calculated:

59.69% C 6.05 _0/0 H 0/0 11.60 N, 6.64% S; found:

59.49% C 6.15% H 11.52 0 / N ₀ 6.58 _0/0 S

Amino Acid Analysis: Cys (Bzl) 1.03, Tyr 0.97, Phe 1.04, Glu 1.00, Asp 0.98, Pro 1.03, Arg 0.95.

3-Mercaptopropionyl-tyrosyl-phenylalanyl-glutaminyl-asparaginyl-cysteinyl-prolyl-D-arginine cyclic disulfide

The protected octapeptide prepared above (100 milligrams) was dissolved with stirring in liquid ammonia (300 ml) and reduced with sodium for 15 seconds. After addition of NH 4 Cl, the ammonia was evaporated, the residue dissolved with cooling in 0.01 M HCl (200 ml). The pH was adjusted to 6.75. The solution containing the linear peptide of formula (II) was extracted with ether, brought to a volume of 300 ml and a solution of 0.01 M potassium ferrocyanide was added dropwise to a permanent yellow color. The solution was then filtered through a weakly basic anion exchange column in a C1-cycle, the eluates were lyophilized, and the crude product was purified by gel filtration. 10 mg of pure product of [α] _D -56.7 ° (c 0.1, 1M acetic acid) was obtained. R _F 0.32 (2-butanol-98% formic acid-water, 75: 13.5: 11.5), 0.54 (1-butanol-acetic acid-pyridine-water, 15: 3: 10: 6), 0.71 (1-butanol-acetic acid-ethyl acetate-water, 1: 1: 1: 1), 0.71 (ethyl acetate-pyridine-acetic acid-water, 5: 5: 1: 3) ). The product was electrophoretically pure in two buffers of different values.

For C44H60N12O12S2. C2H4O2. 2H2O (1109). calculated:

49.81 0/0 C, 6.18 O / o H, 15.15 «/ o N, found:

H, 5.97; N, 15.09.

Amino Acid Analysis: Tyr 0.96, Phe 1.06, Glu 1.03, Asp 0.98, Pro 1.02, Arg 0.94.

Claims (2)

VYNALEZU A v-asopressin analogue of formula I, S ------ S CH, 1 CH 2 CO-Tyr-Ph & -Gin-Asn-NH-C. Where all the amino acids are L-series, the casein of formula I, characterized in that the league arginine is a D-series. a non-linear peptide of formula II 2. A method for producing a novel vasoSH SH analog I I CH2 CH2 CH2-CO-Tyr-Phe-Gin-Asn-NH-CH-CO-Pro-Arg-OH oxidizes to form a disulfide bond.