CS226923B1 - Xytocine analoggues with inhibiting efects and method offreparing same - Google Patents

Description

The present invention provides oxytocene analogues having an inhibitory effect containing a aromatic amino acid of the D-configuration at position 2 and a process for their preparation.

It is known that neurohypophyseal hormones have a number of biological activities for which they are used in medicine. In certain cases, an agent having an inhibitory effect on any of the activities would be appropriate. They could find use, for example, in various complications during pregnancy and during labor.

It has now been found that oxytocin analogs containing at position 2 the phenylalanine or p-position substituted phenylalanine moiety having the D configuration have the ability to inhibit the uterotonic activity of oxytocin. Accordingly, the present invention provides oxytocin analogs of the invention having the general formula (I)

2? £

R - C - * - R - (jH *

R-C HC ON HC H-CO-Ile-GLa-Asrx-NH-CH-CO-Pro-u-GLy-NH _from CH _Z (I) where R ¹ and R ² is H or CH 3, R ³ is H or NH2, ^R4 is H or alkyl of one to three carbons or alkoxy of 1-2 carbons, R ⁵ is a group s-s or s-CH _2, the amino acid is D-configuration and the other chiral amino acids have L configuration

The essence of the process for the preparation of the novel oxytocin analogs of formula I, wherein R ⁵ is S-S according to the invention, is to produce a linear

SH

R-C-t

CH,

R ~ CH ~ CO ~ NH ~ CH ~ CO ~ ILe ~ GLt2 ~ Azri ~ N H-CH-CO — Pro-Leu-GL ^ -NH ^

(II), wherein R ¹ -R ⁴ have the same meaning as in Formula I, oxidizes to form a disulfide bond. Another possible method of making the analogs of the invention is that of linear peptide of formula III

I ·

Where R ⁴ has the same meaning as in Formula I, it undergoes cyclizations to form a peptide bond between the amino group of the aromatic amino acid and the carboxyl group of the S-carboxyethylhomocysteine residue.

The pA ₂ values for some of the prepared compounds in the rat uterine test are given in (Table I. The pA ₂ value corresponds to the negative logarithm of the inhibition constant according to the equation where B is the inhibitor concentration (M), A ₅₀ is the agonist concentration leading to 50% and 50 50 _µl is the concentration of agonism resulting in a 50% maximal effect in the presence of inhibitor B (Eggena, P., Schwartz, IL, Walt, R .: J. Gen. Physiol, 52, 465 (1968)).

pA ₂ = -log _B

A50B

A50

Table I

PA ₂ values in the rat uterus test

Compound of formula R ¹ R ²

I Η H

I Η H

I Η H

I Η H

I Η H

I Η H

I CH ₃ ch ₃

The preparation of oxytocin analogs is further illustrated in the Examples.

Example 1 [2-D-Phenylalanine-deamino-6-carbo-oxytocin o-Nitrobenzenesulfenyl-D-phenylalanyl-isoleucyl-glutaminyl-asparagiinyl-S- (β-carboxyethyl) homocysteinyl-prolyl-leucyl-glycinamide (150 mg) was dissolved in dimethylformamide (4 ml) and after addition of a solution of hydrogen chloride in ether (3.2 M; 0.4 ml) the resulting mixture was allowed to stand at room temperature for 6 min. Addition of ether precipitated octapeptide hydrochloride, which was precipitated once more from dimethylformamide and ether, suctioned off and dried in vacuo. Pa '6 was dissolved in dimethylformamide (4 ml) and after addition of hydroxybenzotriazole (181 mg) and cooling to 0 ° C, dicyclohexylcarbodiimide (200 mg) was added to the mixture. The solution was stirred for 1 h at 0 ° C and 1.5 h at room temperature. The precipitated urea crystals were aspirated and the solution was taken up in methanol (150 ml) heated to 45 ° C and the pH of the mixture was adjusted to 8.5 by addition of N-ethylpiperidine. The mixture was heated at 45 ° C for 2 h and then concentrated to a small volume. Addition of ether precipitated the product, which was aspirated and dried in vacuo (140 mg). A portion of the product (30 milligrams) was dissolved in a 1: 1 mixture of methanol and water (10 ml) and loaded onto a Partisil ODS-2 column (50 x 0.9 cm).

Elution was carried out with a mixture of 55% methanol with 0.05% aqueous trifluoroacetic acid. The fraction containing k = 7.3 was concentrated in vacuo and lyophilized. 6.5 mg of pure by HPLC and TLC were obtained in four systems.

For C 4 H ₄ O _C7 N _{u u} S. 2 H ₂ O (994.2) was calculated:

% C, 53.16;% H, 7.20; found:

% H, 7.39;% N, 15.29.

Amino Acid Analysis: issp 1.04, Glu 1.05, Pro 0.95, Gly 1.04, Ile

R ³ R - * R ⁵ pA ₂

H H S — CH, 8.26 H 0C H ₅ S — CH, 7.54 H ch ₃ S — ch ₂ 8.73 H c, h ₅ S — CH, 8.73 H c ₂ h ₅ s — s 8.06 NH _a c ₂ h ₅ s — s 8.15 nh ₃ C ₂ H ₅ s — s 8,09 0,94, Leu 1.05, Phe 0.98, Hcy (0 _{2 4} Η ΟΟΟΗ)

0.97.

Example 2 [2-D-O-Ethyltyrosine] deamino-6-carbo-oxytocin o-Nitrobenzenesulfenyl-DO-ethyltyrosyl-isoleucyl-glutaminyl-asparaginyl-S- (S-carboxyethyl) homocysteimyl-prolyl-leucyl-glycinamide ( 130 mg) was cyclized in the same manner as in Example 1. 135 mg of crude product was obtained, a portion (30 milligrams) of which was dissolved in methanol-water (4: 6) and loaded onto a Partisil ODS-2 column (50 x 0, 9 cm). Elution was performed with a mixture of methanol and 0.05% trifluoroacetic acid (1: 1). The fraction containing ok-26.3 was concentrated and lyophilized. 6.2 mg of pure by HPLC and TIC were obtained.

For C 11 H 11 N 11 O 2 S. 4 H ₂ O (1074) calculated:

% C, 51.43;% H, 7.41;% N, 14.34; found:

% C, 51.27;% H, 7.65;

Amino acid analysis:

Asp 1.02, Gen 1.03, Pro 1.00, Gly 1.04, Ile

0.93, Leu 0.98, Tyr (Et) 0.41, Tyr 0.56, Hcy (C ₂ H ₄ COOH) 0.92.

EXAMPLE 3 [2-Dp-Methylphenylalanine] Deamino-6-Carboxytocin o-Niltrobenzenesulfeinyl-Dp-methylphenylalnnyl-isoleucyl-glutaminyl-aspariaginyl-S- (β-carboxyyeethyl) homocysteinyl-prolyl-leucyl-glycinamide (220 mg) A portion of the crude product (30 mg) was purified in the same manner as in Example 1. The fraction containing mesh = 13.6 was concentrated in vacuo and lyophilized. 7.3 mg of pure by HPLC and TLC were obtained.

22S923

For C ₄₅ H ₆₉ N _X1 O _U N. 3 H ₂ 0 (1026) calculated:

% H, 7.37;% N, 15.01; nelezeno:

% C, 52.40;% H, 7.61;% N, 14.83.

Amino acid analysis:

Asp 1.00, Glu 1.00, Pro 1.05, Gly 1.01, Ile

0.91, Leu 1.04, Phe (Ma) 0.99, Hcy (C ₂ H ₄ COOH) 0.99.

Example 4 [2-Dp-Ethyl-phenylalanine] deamino-6-carbo-oxytocin o-Nitrobenzenesulphenyl-D-ethylphenylalanyl-isoleucyl-glutaminyl-asparaginyl-S- (β-carboxyethyl) homocysteinyl-prolyl-leucyl-glyclmamide ( 200 mg) was cyclized and a portion (30 mg) of the product was purified as in Example 1. 5.3 mg of pure by HPLC and TLC were obtained.

For C ₄₆ H ₇₁ N _u O _u S. 4 H ₂ O (1058) was calculated:

% C, 52.21;% H, 7.52;% N, 14.56; found:

% C, 51.90;% H, 7.54;% N, 14.41.

Amino acid analysis:

Asp 1.01, Glu 1.03, Pro 0.94, Gly 0.99, Ile

0.92, Leu 1.05, Phe (Et) 1.00, Hcy (C ₂ H ₄ COOH) 0.95.

EXAMPLE 5 [2-D-p-Ethylphenylalanine] Deamino-oxytocin

S-Benzylmercaptopropionyl-Dp-ethylphenylalanyl-isoleucyl-glutaminyl-asparaginyl-S-benzylcysteinyl-prolyl-leucyl-glycinamide (50 mg) was dissolved in liquid ammonia and reduced with a sodium bar until the blue color was stable for 1 minute. The blue color was removed by adding a drop of acetic acid and the mixture was lyophilized. The lyophilisate was dissolved in 0.1 M HC1 (8 ml), the solution was diluted to 100 mL with water and the addition of 0.1 M NaOH, pH was adjusted to 7. Then was added over 20 minutes a solution of K ₃ Fe (CN) ₆ (25 mg in 5 ml of water) while maintaining the pH at 7. The mixture was stirred at room temperature for 40 minutes. The pH was then adjusted to 4 by the addition of acetic acid and the solution was loaded by high pressure pump onto a Separon SI-C-18 column (25 x 0.4 centimeters). The column was washed with water and the peptides eluted with a mixture of 80% methanol and 0.05% aqueous trifluoroacetic acid. The eluate was concentrated in vacuo and lyophilized. The lyophilisate was redissolved in methanol / water (1: 1; 8 mL), loaded onto a Partisil ODS-2 column (50 x 0.9 cm) and eluted with methanol: 0.05% trifluoroacetic acid (65:35). The fraction containing mesh = 16.6 was concentrated in vacuo and lyophilized. 8.6 mg of pure by HPLC and TLC were obtained.

For C ₄₅ H ₆₉ N _XX O _X1 S ₂ . 4 H ₂ O (1076) was calculated:

H, 7.21; N, 14.32; found:

H, 7.24; N, 14.18.

Amino acid analysis:

Asp 1,02 ,. Glu 0.97, Pro 0.89, Gly 1.00, Cys

0.53, Ile 0.91, Leu 1.00, Phe (Et) 1.00. Example 6 [2-D-p-Ethyl-phenylalanine] oxytocin

N -Benzyloxycarbomyl-S-benzylcysteinyl-Dp-ethylphenylalanyl-isoleucyl-glutaninyl-aspiaraginyl-S-benzylcysteinyl-prolyl-leucyl-glycinamide (200 mg) was reduced and oxidized as in Example 5. The cyclization solution was loaded on an Amberlite CG column. 50 (30 mL), the column was washed with 0.25% acetic acid and the peptides eluted with 50% acetic acid. A portion of the product obtained by lyophilization (1/3) was dissolved in a 1: 1 mixture of methanol and water (8 ml) and loaded onto a Fartisil ODS-2 column (50 x 0.9 cm). Elution was performed with a mixture of methanol (60%) with 0.1 M ammonium acetate (40%), pH 7. The fraction containing k = 21.1 was concentrated in vacuo and lyophilized. It was obtained. 16.3 mg pure by HPLC and TLC.

For C ₄₅ H ₇₀ N O _{XX X 2 second} 2 H ₂ O. C ₂ H ₄ O ₂ (1115) was calculated:

% C, 50.61;% H, 7.05;% N, 15.07; found:

% C, 50.36;% H, 6.81;% N, 15.23.

Amino acid analysis:

Asp 0.96, Glu 0.98, Pro 1.02, Gly 0.96, Cys

1.73, Ile 1.06, Leu 1.03, Phe (Et) 1.02.

Example 7 [1-Peuicillamine-2-D-p-ethylphenylalanine] oxytocin

N ^K -Benzyloxycarbonyl-S-benzylpenicilaminyl-Dp-ethylphenylalanyl-isoleucyl-glutaminyl-asparaginyl-S-benzylcysteinyl-prolyl-leucyl-glycinamide (200 mg) was reduced and oxidized in the same manner as in Example 5. The cyclization mixture was treated and purified in the same manner. as in Example 6, except that a mixture containing 65% methanol was used for elution. 8.2 mg of pure by HPLC and TLC were obtained.

2 ¢ -9.2 .3

For C ₄₇ H ₇₄ N ₁₂ OhS ₂ .3 H ₂ O. C ₂ H ₄ O ₃ (1161) Amino acid analysis:

Asp was 0.98, Glu 1.00, Pro 0.89, Gly 1.00, Ile

1.03, Leu 1.02 1.02, Phe (Et) 0.97, Cys + calculated: + δθ 1.48.

C, 50.67; H, 7.29; N, 14.47;

found:

% C, 50.78;% H, 7.43;% N, 14.31.

Claims (3)

I will invent the subject An oxytocin analogue of the formula I wherein R ¹ and R ² is H or CH 3, R ³ is H or NH 2, R ⁴ is H or alkyl of one to three carbons or alkoxy of 1 to 2 carbons, R ⁵ is S-S or S-CH ₂ , the amino acid marked with an asterisk is the D-configuration, and the other chiral amino acids are of the L configuration. 2. A process for the preparation of oxytocin analogs of formula (I) wherein R < ^{5 &gt}; is S-S- and the other substituents are as defined in (1), characterized in that the linear peptide of formula (II) SH CH? SH RCR ¹ 31 * R-CH-CO-NH-CH-CO-Ile-Gln-Azn-NH-CH-CO-Pro-Leu-Gly-NH? (II) wherein R ¹ to R ⁴ have the same meaning as in formula I, oxidizes to form a disulfide bond. 3. A process for producing an analog of oxytocin obec12 Joint formula I wherein R ⁵ has the meaning of S-CH ₂ and the other substituents have the meaning given point 1, characterized in that a linear peptide of Formula III -O Πλ NH-CH-C0-Pro-LeurGLy-N1 · t where R ⁴ has the same meaning as in Formula I, undergoes cyclization to form a peptide bond between the amino group of the aromatic amino acid and the carboxyl group of the S-carboxyethylhomocysteine residue.